https://proteopedia.org/api.php?action=feedcontributions&feedformat=atom&user=Judy+VoetProteopedia - User contributions [en]2026-09-20T06:03:14ZUser contributionsMediaWiki 1.43.8https://proteopedia.org/index.php?title=User:Judy_Voet&diff=2530230User:Judy Voet2016-02-07T04:40:51Z<p>Judy Voet: </p>
<hr />
<div>[[Image:Judy Voet.jpg]]<br />
{{Clear}}<br />
J. H. Hammons Professor, Emeritus, Chem/Biochem Dept, Swarthmore College<br />
Co-Editor-in Chief, Biochemistry and Molecular Biology Education 2000-2014<br />
<br />
Co-Author, Biochemistry, 4th Ed<br />
Co-Author Fundamentals of Biochemistry 5th Ed</div>Judy Voethttps://proteopedia.org/index.php?title=Hen_Egg-White_(HEW)_Lysozyme&diff=1132500Hen Egg-White (HEW) Lysozyme2010-10-13T01:54:55Z<p>Judy Voet: </p>
<hr />
<div><applet load='aln_1H6M_to_1HEW_2.pdb' size='300' frame='true' align='right' caption=' Hen Egg White (HEW) Lysozyme containing a trisaccharide of N-acetylglucosamine (NAG) bound to the active site, PDBid 1HEW' scene='User:Judy_Voet/Lysozyme/Lysozyme1/16' /><br />
Lysozyme was the first enzyme whose X-ray structure was determined <ref> PMID 5840126</ref><ref>Phillips, D. C. The hen egg white lysozyme molecule. Proc. Natl Acad. Sci. USA 57, 483-495 (1967)</ref>. This <scene name='User:Judy_Voet/Lysozyme/Lysozyme1/15'>scene </scene> shows Hen Egg White (HEW) lysozyme containing a trisaccharide of N-acetylglucosamine (NAG) bound to a cleft in the enzyme. David Phillips, who determined the structure in 1965, saw that the cleft was large enough to fit three more saccharide units. He therefore built a model extending the trisaccharide to a <br />
<scene name='User:Judy_Voet/Lysozyme/Lysozyme1_hexamer/7'>hexasaccharide</scene> that fits into the cleft, labeling the sugar subsites A-F<ref> coordinates of the model kindly provided by Louise Johnson</ref>. Alternately click on <scene name='User:Judy_Voet/Lysozyme/Lysozyme1/15'>trisaccharide</scene> and <scene name='User:Judy_Voet/Lysozyme/Lysozyme1_hexamer/7'>hexasaccharide</scene> to turn the modeled portion of the hexasaccharide on and off.<br />
The interesting thing about the model was that the only way that the hexasaccharide would fit into the cleft was if the 4th saccharide (in subsite D) was strained into a <scene name='User:Judy_Voet/Lysozyme/Half-chair/2'>half-chair conformation</scene>. This conformation is what would be necessary for the formation of an oxocarbenium ion (oxionium ion). When the model was studied, <scene name='User:Judy_Voet/Lysozyme/Glu_35/1'>Glu 35</scene> was found to be in an ideal location to act as a general acid catalyst, 3.34 Angstroms from the bridging oxygen between the 4th and 5th saccharide units. <scene name='User:Judy_Voet/Lysozyme/Asp_52/2'>Asp 52</scene> appeared to be too far away (2.69 angstroms) in the static lysozyme structure to have formed a covalent bond with C1 of the half-chair model in the D site, and no covalent intermediate had ever been detected, so Phillips proposed that it acted as an electrostatic stabilizer of the oxonium ion (referred to as The Phillips Mechanism).<br />
<br />
{{Clear}}<br />
<br />
<applet load='aln_1H6M_to_1HEW_2.pdb' size='300' frame='true' align='right' caption='NAG-2-deoxy-2-fluoro-glucosyl fluoride (NAG2FGlcF) bound to Glu35Gln HEW Lysozyme PDBid 1H6M' scene='User:Judy_Voet/Lysozyme/1h6m/3'/><br />
Then, in 2001, Stephen Withers published <scene name='User:Judy_Voet/Lysozyme/1h6m/3'>1H6M</scene>,<ref>PMID 11518970</ref> in which Glu 35 had been mutated to Gln to remove the general acid catalyst. The substrate contained NAG-2-fluoro-glucosyl fluoride (NAG2FGlcF). The fluoro group on C-1 does not require acid catalysis to be a good leaving group, and the remaining saccharide, in the absence of the acid necessary to catalyse the second step of the reaction, was demonstrated to form a <scene name='User:Judy_Voet/Lysozyme/Covalent/1'> covalent intermediate</scene>. In this <scene name='User:Judy_Voet/Lysozyme/Superposition/2'>superposition</scene> of the half chair model with 1HEW (greens) and the covalent intermediate in 1H6M (blues), note the relatively small motions of Asp 52 and C1 of the sugar ring in going from the model to the covalent intermediate. to observe the motion from the <scene name='User:Judy_Voet/Lysozyme/Asp52_halfchair/1'>half-chair</scene> to the <scene name='User:Judy_Voet/Lysozyme/Covalent/2'>covalent intermediate</scene> just toggle between the two green links. <br />
===Some Useful External Links===<br />
[http://en.wikipedia.org/wiki/Lysozyme Lysozyme]<br />
<br />
[http://en.wikipedia.org/wiki/Glycoside_hydrolase#Retaining_glycoside_hydrolases Retaining Glycoside Hydrolases]<br />
===References===<br />
<references/></div>Judy Voethttps://proteopedia.org/index.php?title=Phosphofructokinase_(PFK)&diff=1020116Phosphofructokinase (PFK)2009-11-22T20:45:17Z<p>Judy Voet: /* External Links */</p>
<hr />
<div>==Overview==<br />
Phosphofructokinase-1 (PFK-1) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, PEP and fructose-2,6-bisphosphate (F2,6P). <br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<scene name='Sandbox/Judy_Voet/PFK/4pfk_biol/1'>PFK from B. stearothermophilus</scene> is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_overview/2'>subunits A (light Blue) and D (yellow)</scene> with the active site interfaces in magenta with the substrates in cyan. Two more active sites are at the interface of subunits B (green) and C (pink). A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/2'>A/D interface active site</scene> of subunit D (Yellow) shows that amino acids from both subunits A (light blue) and D (Yellow) contribute to the binding of F6P. Two of the allosteric sites are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_overview/2'>subunits A and B</scene> and two at the interface of subunits C and D. Again the interfaces are magenta with the allosteric ligand in cyan. A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_closeup/4'>A/B allosteric site</scene> of subunit A shows contributions from both subunits to the binding of ADP. The conformational changes in going between the R and T states of PFK are illustrated below.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
== Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1: PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are part of the F6P binding site in the T and R states, srespectively(see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2: Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three residues (2 arg and 1 Lys; not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1: The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the absence of the positive charge of Arg 162 have on the binding of F6P? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2: Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
At one time, the negative charge of Glu 161 was thought to have a negative effect on F6P binding in the T state. This idea has not been supported by site-directed mutagenesis experiments<ref>PMID:10759544</ref>.Several mutant PFKs have been made, including R162A, E161A and R162A/E161A. The R162A mutation caused a 30-fold decrease in F6P binding. The E161A mutation, however, had little effect on the ability of PEP to inhibit F6P binding. <br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/><br />
==External Links==<br />
[http://www.pdb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb50_4.html PDB Molecule of the Month PFK]<br />
[http://en.wikipedia.org/wiki/Phosphofructokinase_1 Wikipedia Phosphofructokinase_1]<br />
<br />
<br />
[[Category:glycolysis]]</div>Judy Voethttps://proteopedia.org/index.php?title=Phosphofructokinase_(PFK)&diff=1020112Phosphofructokinase (PFK)2009-11-22T20:35:47Z<p>Judy Voet: /* External Links */</p>
<hr />
<div>==Overview==<br />
Phosphofructokinase-1 (PFK-1) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, PEP and fructose-2,6-bisphosphate (F2,6P). <br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<scene name='Sandbox/Judy_Voet/PFK/4pfk_biol/1'>PFK from B. stearothermophilus</scene> is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_overview/2'>subunits A (light Blue) and D (yellow)</scene> with the active site interfaces in magenta with the substrates in cyan. Two more active sites are at the interface of subunits B (green) and C (pink). A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/2'>A/D interface active site</scene> of subunit D (Yellow) shows that amino acids from both subunits A (light blue) and D (Yellow) contribute to the binding of F6P. Two of the allosteric sites are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_overview/2'>subunits A and B</scene> and two at the interface of subunits C and D. Again the interfaces are magenta with the allosteric ligand in cyan. A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_closeup/4'>A/B allosteric site</scene> of subunit A shows contributions from both subunits to the binding of ADP. The conformational changes in going between the R and T states of PFK are illustrated below.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
== Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1: PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are part of the F6P binding site in the T and R states, srespectively(see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2: Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three residues (2 arg and 1 Lys; not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1: The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the absence of the positive charge of Arg 162 have on the binding of F6P? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2: Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
At one time, the negative charge of Glu 161 was thought to have a negative effect on F6P binding in the T state. This idea has not been supported by site-directed mutagenesis experiments<ref>PMID:10759544</ref>.Several mutant PFKs have been made, including R162A, E161A and R162A/E161A. The R162A mutation caused a 30-fold decrease in F6P binding. The E161A mutation, however, had little effect on the ability of PEP to inhibit F6P binding. <br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/><br />
==External Links==<br />
[http://www.pdb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb50_4.html PDB Molecule of the Month PFK]<br />
[http://en.wikipedia.org/wiki/Phosphofructokinase_1 Phosphofructokinase_1]<br />
<br />
<br />
[[Category:glycolysis]]</div>Judy Voethttps://proteopedia.org/index.php?title=Phosphofructokinase_(PFK)&diff=1020111Phosphofructokinase (PFK)2009-11-22T20:32:01Z<p>Judy Voet: </p>
<hr />
<div>==Overview==<br />
Phosphofructokinase-1 (PFK-1) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, PEP and fructose-2,6-bisphosphate (F2,6P). <br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<scene name='Sandbox/Judy_Voet/PFK/4pfk_biol/1'>PFK from B. stearothermophilus</scene> is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_overview/2'>subunits A (light Blue) and D (yellow)</scene> with the active site interfaces in magenta with the substrates in cyan. Two more active sites are at the interface of subunits B (green) and C (pink). A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/2'>A/D interface active site</scene> of subunit D (Yellow) shows that amino acids from both subunits A (light blue) and D (Yellow) contribute to the binding of F6P. Two of the allosteric sites are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_overview/2'>subunits A and B</scene> and two at the interface of subunits C and D. Again the interfaces are magenta with the allosteric ligand in cyan. A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_closeup/4'>A/B allosteric site</scene> of subunit A shows contributions from both subunits to the binding of ADP. The conformational changes in going between the R and T states of PFK are illustrated below.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
== Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1: PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are part of the F6P binding site in the T and R states, srespectively(see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2: Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three residues (2 arg and 1 Lys; not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1: The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the absence of the positive charge of Arg 162 have on the binding of F6P? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2: Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
At one time, the negative charge of Glu 161 was thought to have a negative effect on F6P binding in the T state. This idea has not been supported by site-directed mutagenesis experiments<ref>PMID:10759544</ref>.Several mutant PFKs have been made, including R162A, E161A and R162A/E161A. The R162A mutation caused a 30-fold decrease in F6P binding. The E161A mutation, however, had little effect on the ability of PEP to inhibit F6P binding. <br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/><br />
==External Links==<br />
[http://www.pdb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb50_4.html PDB Molecule of the Month PFK]<br />
[http://en.wikipedia.org/wiki/Phosphofructokinase_1 Phosphofructokinase_1]<br />
<br />
<br />
[[Category:glycolysis]]<br />
[[Category:Kinases]]<br />
[[Category: Glycolysis enzymes]]<br />
[[Category:EC 2.7.1]]</div>Judy Voethttps://proteopedia.org/index.php?title=Phosphofructokinase_(PFK)&diff=1020110Phosphofructokinase (PFK)2009-11-22T20:27:14Z<p>Judy Voet: /* External Links */</p>
<hr />
<div>==Overview==<br />
Phosphofructokinase-1 (PFK-1) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, PEP and fructose-2,6-bisphosphate (F2,6P). <br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<scene name='Sandbox/Judy_Voet/PFK/4pfk_biol/1'>PFK from B. stearothermophilus</scene> is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_overview/2'>subunits A (light Blue) and D (yellow)</scene> with the active site interfaces in magenta with the substrates in cyan. Two more active sites are at the interface of subunits B (green) and C (pink). A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/2'>A/D interface active site</scene> of subunit D (Yellow) shows that amino acids from both subunits A (light blue) and D (Yellow) contribute to the binding of F6P. Two of the allosteric sites are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_overview/2'>subunits A and B</scene> and two at the interface of subunits C and D. Again the interfaces are magenta with the allosteric ligand in cyan. A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_closeup/4'>A/B allosteric site</scene> of subunit A shows contributions from both subunits to the binding of ADP. The conformational changes in going between the R and T states of PFK are illustrated below.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
== Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1: PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are part of the F6P binding site in the T and R states, srespectively(see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2: Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three residues (2 arg and 1 Lys; not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1: The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the absence of the positive charge of Arg 162 have on the binding of F6P? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2: Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
At one time, the negative charge of Glu 161 was thought to have a negative effect on F6P binding in the T state. This idea has not been supported by site-directed mutagenesis experiments<ref>PMID:10759544</ref>.Several mutant PFKs have been made, including R162A, E161A and R162A/E161A. The R162A mutation caused a 30-fold decrease in F6P binding. The E161A mutation, however, had little effect on the ability of PEP to inhibit F6P binding. <br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/><br />
==External Links==<br />
[http://www.pdb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb50_4.html PDB Molecule of the Month PFK]<br />
[http://en.wikipedia.org/wiki/Phosphofructokinase_1 Phosphofructokinase_1]<br />
<br />
<br />
{{glycolysis}}<br />
{{Kinases}}<br />
{{Glycolysis enzymes}}<br />
[[Category:EC 2.7.1]]</div>Judy Voethttps://proteopedia.org/index.php?title=Phosphofructokinase_(PFK)&diff=1020106Phosphofructokinase (PFK)2009-11-22T20:16:06Z<p>Judy Voet: /* External Links */</p>
<hr />
<div>==Overview==<br />
Phosphofructokinase-1 (PFK-1) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, PEP and fructose-2,6-bisphosphate (F2,6P). <br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<scene name='Sandbox/Judy_Voet/PFK/4pfk_biol/1'>PFK from B. stearothermophilus</scene> is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_overview/2'>subunits A (light Blue) and D (yellow)</scene> with the active site interfaces in magenta with the substrates in cyan. Two more active sites are at the interface of subunits B (green) and C (pink). A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/2'>A/D interface active site</scene> of subunit D (Yellow) shows that amino acids from both subunits A (light blue) and D (Yellow) contribute to the binding of F6P. Two of the allosteric sites are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_overview/2'>subunits A and B</scene> and two at the interface of subunits C and D. Again the interfaces are magenta with the allosteric ligand in cyan. A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_closeup/4'>A/B allosteric site</scene> of subunit A shows contributions from both subunits to the binding of ADP. The conformational changes in going between the R and T states of PFK are illustrated below.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
== Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1: PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are part of the F6P binding site in the T and R states, srespectively(see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2: Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three residues (2 arg and 1 Lys; not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1: The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the absence of the positive charge of Arg 162 have on the binding of F6P? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2: Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
At one time, the negative charge of Glu 161 was thought to have a negative effect on F6P binding in the T state. This idea has not been supported by site-directed mutagenesis experiments<ref>PMID:10759544</ref>.Several mutant PFKs have been made, including R162A, E161A and R162A/E161A. The R162A mutation caused a 30-fold decrease in F6P binding. The E161A mutation, however, had little effect on the ability of PEP to inhibit F6P binding. <br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/><br />
==External Links==<br />
[http://www.pdb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb50_4.html PDB Molecule of the Month PFK]<br />
[http://en.wikipedia.org/wiki/Phosphofructokinase_1 Phosphofructokinase_1]</div>Judy Voethttps://proteopedia.org/index.php?title=Phosphofructokinase_(PFK)&diff=1020105Phosphofructokinase (PFK)2009-11-22T20:15:22Z<p>Judy Voet: /* External Links */</p>
<hr />
<div>==Overview==<br />
Phosphofructokinase-1 (PFK-1) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, PEP and fructose-2,6-bisphosphate (F2,6P). <br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<scene name='Sandbox/Judy_Voet/PFK/4pfk_biol/1'>PFK from B. stearothermophilus</scene> is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_overview/2'>subunits A (light Blue) and D (yellow)</scene> with the active site interfaces in magenta with the substrates in cyan. Two more active sites are at the interface of subunits B (green) and C (pink). A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/2'>A/D interface active site</scene> of subunit D (Yellow) shows that amino acids from both subunits A (light blue) and D (Yellow) contribute to the binding of F6P. Two of the allosteric sites are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_overview/2'>subunits A and B</scene> and two at the interface of subunits C and D. Again the interfaces are magenta with the allosteric ligand in cyan. A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_closeup/4'>A/B allosteric site</scene> of subunit A shows contributions from both subunits to the binding of ADP. The conformational changes in going between the R and T states of PFK are illustrated below.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
== Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1: PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are part of the F6P binding site in the T and R states, srespectively(see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2: Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three residues (2 arg and 1 Lys; not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1: The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the absence of the positive charge of Arg 162 have on the binding of F6P? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2: Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
At one time, the negative charge of Glu 161 was thought to have a negative effect on F6P binding in the T state. This idea has not been supported by site-directed mutagenesis experiments<ref>PMID:10759544</ref>.Several mutant PFKs have been made, including R162A, E161A and R162A/E161A. The R162A mutation caused a 30-fold decrease in F6P binding. The E161A mutation, however, had little effect on the ability of PEP to inhibit F6P binding. <br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/><br />
==External Links==<br />
[http://www.pdb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb50_4.html PDB Molecule of the Month PFK]<br />
[http://en.wikipedia.org/wiki/Phosphofructokinase_1 Phosphofructokinase_1]</div>Judy Voethttps://proteopedia.org/index.php?title=Phosphofructokinase_(PFK)&diff=1020104Phosphofructokinase (PFK)2009-11-22T20:12:22Z<p>Judy Voet: </p>
<hr />
<div>==Overview==<br />
Phosphofructokinase-1 (PFK-1) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, PEP and fructose-2,6-bisphosphate (F2,6P). <br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<scene name='Sandbox/Judy_Voet/PFK/4pfk_biol/1'>PFK from B. stearothermophilus</scene> is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_overview/2'>subunits A (light Blue) and D (yellow)</scene> with the active site interfaces in magenta with the substrates in cyan. Two more active sites are at the interface of subunits B (green) and C (pink). A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/2'>A/D interface active site</scene> of subunit D (Yellow) shows that amino acids from both subunits A (light blue) and D (Yellow) contribute to the binding of F6P. Two of the allosteric sites are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_overview/2'>subunits A and B</scene> and two at the interface of subunits C and D. Again the interfaces are magenta with the allosteric ligand in cyan. A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_closeup/4'>A/B allosteric site</scene> of subunit A shows contributions from both subunits to the binding of ADP. The conformational changes in going between the R and T states of PFK are illustrated below.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
== Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1: PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are part of the F6P binding site in the T and R states, srespectively(see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2: Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three residues (2 arg and 1 Lys; not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1: The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the absence of the positive charge of Arg 162 have on the binding of F6P? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2: Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
At one time, the negative charge of Glu 161 was thought to have a negative effect on F6P binding in the T state. This idea has not been supported by site-directed mutagenesis experiments<ref>PMID:10759544</ref>.Several mutant PFKs have been made, including R162A, E161A and R162A/E161A. The R162A mutation caused a 30-fold decrease in F6P binding. The E161A mutation, however, had little effect on the ability of PEP to inhibit F6P binding. <br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/><br />
==External Links==<br />
Wikipedia [http://www.pdb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb50_4.html]<br />
PDB Molecule of the Month [http://en.wikipedia.org/wiki/Phosphofructokinase_1]</div>Judy Voethttps://proteopedia.org/index.php?title=Proteopedia:Table_of_Contents&diff=1020094Proteopedia:Table of Contents2009-11-22T19:12:37Z<p>Judy Voet: </p>
<hr />
<div>{{#tree:id=siteTree|openlevels=1|root='''Chemistry of Life'''|close=top|open=top|<br />
<br />
* INTRODUCTION<br />
** Introduction to the Chemistry of Life<br />
<br />
** WATER<br />
<br />
*** [[Water_in_macromolecular_models|Water in Macromolecular Models]]<br />
** KEY CHEMICAL CONCEPTS FOR STRUCTURAL BIOLOGY<br />
<br />
*** [[Cation-pi interactions]]<br />
*** [[Hydrogen bond]]<br />
*** [[Hydrogen_in_macromolecular_models|Hydrogen in Macromolecular Models]]<br />
*** [[Isoelectric_point]]<br />
<br />
* BIOMOLECULES<br />
**[[About_Macromolecular_Structure]]<br />
<br />
** NUCLEOTIDES, NUCLEIC ACIDS AND GENETIC INFORMATION<br />
*** DNA<br />
**** [[DNA]]<br />
**** B-DNA [[1bna]]<br />
**** [[Z-DNA]]<br />
<br />
** AMINO ACIDS<br />
*** [[Amino_Acids]]<br />
*** [[Selenocysteine]]<br />
** PROTEINS: PRIMARY STRUCTURE<br />
*** [[Conservation,_Evolutionary]]<br />
*** [[Isoelectric_point]]<br />
<br />
<br />
** PROTEINS: THREE-DIMENSIONAL STRUCTURE<br />
*** [[S347/The four tiers of protein structure]]<br />
*** [[Secondary_structure]]<br />
*** Protein structural motifs<br />
**** [[User:James_D_Watson/Structural_Templates]]<br />
*** FIBROUS PROTEINS<br />
**** [[Coiled_coil]]<br />
**** [[Collagen]]<br />
*** Protein Misfolding<br />
**** [[Prion_protein]]<br />
**** [[A_Physical_Model_of_the_Structure_of_GNNQQNY_from_Yeast_Prion_Sup35]]<br />
<br />
** COLORED PROTEINS & THOSE THAT CHANGE COLOR<br />
*** [[Green Fluorescent Protein]]; [[GFP_(Hebrew)]]<br />
*** [[Dronpa]]<br />
*** [[Factor IX]]<br />
<br />
** DNA BINDING PROTEINS<br />
*** [[Helix-turn-helix motif]]<br />
*** [[DNA-binding protein VirE2 from Agrobacterium tumefaciens complexed with chaperone VirE1]]<br />
<br />
** PROTEIN FUNCTION: Myoglobin and Hemoglobin, Muscle Contraction, and Antibodies<br />
*** Antibodies<br />
**** [[IgA]]<br />
**** [[Epitopes]]<br />
**** [[Major_Histocompatibility_Complex_Class_I]]<br />
*** [[Calmodulin_in_motion]]<br />
*** [[Myoglobin]]<br />
*** [[Hemoglobin]]; [[Hemoglobin_(Hebrew)]]<br />
*** [[Kinesin-5]]<br />
<br />
** LIPIDS AND BIOLOGICAL MEMBRANES<br />
<br />
** MEMBRANE TRANSPORT PROTEINS<br />
*** [[A Physical Model of the β2-Adrenergic Receptor]]<br />
*** [[Lactose_Permease]]<br />
*** [[Proton_Channels]]<br />
*** [[Ion_channels]]<br />
*** [[Mechanosensitive_channels:_opening_and_closing]]<br />
***[[Enzyme_I_of_the_Phosphoenolpyruvate:Sugar_Phosphotransferase_System]]<br />
<br />
** PRIONS AND INTRINSICALLY DISORDERED PROTEINS<br />
*** [[Prion_protein]]<br />
*** [[Doppel]]<br />
*** [[Intrinsically Disordered Protein]]<br />
<br />
** TOXINS<br />
*** [[Insecticidal delta-endotoxin Cyt2Ba from Bacillus thuringiensis]]<br />
<br />
** MITOSIS, MEOSIS, AND CARGO TRANSPORT PROTEINS<br />
*** [[Kinesin-5]]<br />
<br />
** VIRUSES<br />
*** [[User:Wayne_Decatur/Suppression_of_RNA_Silencing_by_Viruses]]<br />
*** Filamentous bacteriphage<br />
**** [[G3p]] - minor coat protein found on the surface of filamentous bacteriophage<br />
*** HIV<br />
**** [[HIV-1 protease]]<br />
**** [[HIV-1 Gag]]<br />
**** [[HIV-1 Gag Recruitment of Tsg101 and the Viral Budding Process]]<br />
**** [[User:Eric_Martz/Molecular_Playground/HIVDrug]]<br />
*** Herpes Simplex Virus<br />
**** [[Herpes_Simplex_Virus_Thymidine_Kinase]]<br />
*** Influenza<br />
**** [[Influenza_hemagglutinin]]<br />
**** [[Avian_Influenza_Neuraminidase,_Tamiflu_and_Relenza]]<br />
<br />
* ENZYMES<br />
** Enzymatic Catalysis<br />
***[[Hen_Egg-White_%28HEW%29_Lysozyme]]<br />
*** Serine Hydrolases<br />
**** [[Serine_Protease]]<br />
**** [[Trypsin]]<br />
**** [[Alpha-1-antitrypsin]]<br />
**** [[Acetylcholinesterase]] (AChE)<br />
***** [[Acetylcholine]]<br />
***** [[Flexibility_of_aromatic_residues_in_acetylcholinesterase]]<br />
***** [[AChE inhibitors and substrates]]<br />
****** [[1eve]] AChE-Aricept complex; [[1eve (Chinese)]]; [[1eve (Russian)]]; [[1eve (Spanish)]]; [[1eve (Turkish)]]<br />
****** [[AChE_bivalent_inhibitors]]<br />
*** Cysteine Proteases<br />
**** [[Tobacco_Etch_Virus_(TEV)_Protease]]<br />
**** [[Streptomyces_griseus_Aminopeptidase_(SGAP)]]; [[Aminopeptidase]]<br />
*** Acid Proteases<br />
**** [[HIV-1 protease]]<br />
**** [[Pepsin]]<br />
*** Metaloproteases<br />
**** [[Metalloproteases]]<br />
**** [[Matrix_metalloproteinases]]<br />
*** Oxidoreductases<br />
**** [[NADH quinone oxidoreductase ]]<br />
*** [[Triose_Phosphate_Isomerase]]<br />
*** [[Aconitase]]<br />
*** [[Enzyme I of the Phosphoenolpyruvate:Sugar Phosphotransferase System]]<br />
*** [[Pyruvate_phosphate_dikinase]]<br />
***[[Phosphofructokinase_%28PFK%29]]<br />
<br />
** Enzyme Kinetics, Inhibition, and Control<br />
***[[Avian_Influenza_Neuraminidase,_Tamiflu_and_Relenza]]<br />
<br />
** BIOCHEMICAL SIGNALING<br />
*** [[Nitric_oxide_synthase]]<br />
*** [[Recoverin,_a_calcium-activated_myristoyl_switch]]<br />
*** [[Bcl-2]]<br />
*** [[C-Myc]]<br />
<br />
* METABOLISM<br />
** Introduction to Metabolism<br />
<br />
** Glucose Catabolism<br />
*** [[Triose_Phosphate_Isomerase]]<br />
***[[Phosphofructokinase_%28PFK%29]]<br />
<br />
<br />
** Glycogen Metabolism and Gluconeogenesis<br />
*** [[Phosphoglucose_isomerase]]<br />
*** [[Calmodulin_in_motion]]<br />
*** [[Biotin_Protein_Ligase]]<br />
<br />
** Citric Acid Cycle<br />
*** [[Aconitase]]<br />
<br />
** Electron Transport and Oxidative Phosphorylation<br />
*** [[NADH quinone oxidoreductase ]]<br />
<br />
** Photosynthesis<br />
*** [[Photosystem_II]]<br />
*** [[Ribulose-1,5-bisphosphate_carboxylase/oxygenase]]<br />
*** [[PrrA_in_Rhodobacter_sphaeroides]]<br />
*** [[Pyruvate_phosphate_dikinase]]<br />
<br />
** Lipid Metabolism<br />
*** [[Acid-beta-glucosidase]]<br />
** Amino Acid Metabolism<br />
*** [[Aromatic_amino_acid_hydroxylases]]<br />
*** [[Phenylalanine_hydroxylase]]<br />
*** [[Tyrosine_hydroxylase]]<br />
*** [[ATP_Phosphoribosyl_Transferase]]<br />
*** [[Isochorismate_pyruvate_lyase]]<br />
** Mammalian Fuel Metabolism: Integration and Regulation<br />
** Nucleotide Metabolism<br />
*** [[Dihydrofolate_reductase]]<br />
<br />
* GENE EXPRESSION AND REPLICATION <br />
<br />
<br />
** Nucleic Acid Structure<br />
*** [[DNA]]<br />
**** B-DNA [[1bna]]<br />
**** [[Z-DNA]]<br />
*** [[Nucleosomes]]<br />
<br />
** DNA Replication, Repair, and Recombination<br />
*** [[DNA Replication,Transcription and Translation]]<br />
***[[DNA_Polymerase_I]]<br />
*** [[PcrA_helicase]]<br />
*** [[Fpg_Nei_Protein_Superfamily]] - DNA Repair and Base Excision DNA Repair<br />
*** [[Human RecQ-Like protein 1]] - RecQ family of DNA helicases are conserved in from bacteria to man <br />
*** [[Structure_of_E._coli_DnaC_helicase_loader]]<br />
***[[1x9n#Crystal_Structure_of_Human_DNA_Ligase_I_bound_to_5.27-adenylated.2C_nicked_DNA]]<br />
*** [[Rop_protein]]<br />
<br />
** Transcription and RNA Processing<br />
*** [[C-Myc]]<br />
*** [[Lac_repressor]]<br />
*** [[TATA-Binding_Protein]]<br />
*** [[RSP1275]]<br />
*** [[Transcription_Termination_Factor_Rho]]<br />
<br />
** Protein Synthesis<br />
*** [[Ribosome]]<br />
*** [[SelB_Recognition]]<br />
** Regulation of Gene Expression<br />
*** [[Lac_repressor]]<br />
*** [[Irr]]<br />
*** [[P53]]<br />
*** [[Tangible_Models_of_Cdc42_Interacting_With_Intersectin]]<br />
<br />
* EVOLUTION<br />
** [[Conservation, Evolutionary]]<br />
<br />
** [[Extremophiles]]<br />
<br />
* IMMUNE SYSTEM<br />
** Antibodies<br />
*** [[IgA]]<br />
*** [[Epitopes]]<br />
** [[Major_Histocompatibility_Complex_Class_I]]<br />
<br />
<br />
<br />
* METHODS OF STRUCTURE DETERMINATION <br />
** X-ray<br />
*** [[X-ray_crystallography]]<br />
**** [[Asymmetric_Unit]]<br />
**** [[Biological_Unit]]<br />
**** [[Electron_density_maps]]<br />
*** SAXS<br />
** NMR<br />
*** [[NMR_Ensembles_of_Models]]<br />
** Electron Microscopy<br />
<br />
* METHODS OF STRUCTURE & SEQUENCE ANALYSIS <br />
** STRUCTURAL ANALYSIS & VISUALIZATION<br />
*** [[Chime]]<br />
*** Jmol<br />
**** [[S347/Visualising_protein_structure|Visualising Protein Structure]] - Introduction to Jmol<br />
**** [[FirstGlance_in_Jmol]]<br />
*** [[User:Wayne Decatur/Teaching Proteopedia|Teaching Proteopedia]]<br />
*** [[User:Wayne Decatur/Generate Unfolded Structures|Generate Unfolded Structures]]<br />
*** Homology Model<br />
**** [[User:Wayne Decatur/Homology Modeling|Homology Modeling]]<br />
**** [[User:Emi Nakayama/TRIM5a Homology Models|TRIM5a Homology Models]] - Differences in models as a function of their templates<br />
*** [[DRuMS]] - set of standard color schemes for macromolecular visualization<br />
<br />
** SEQUENCE ANALYSIS<br />
*** [[User:Wayne Decatur/Sequence analysis tools|Sequence Analysis Tools]]<br />
<br />
}}</div>Judy Voethttps://proteopedia.org/index.php?title=Proteopedia:Table_of_Contents&diff=1020093Proteopedia:Table of Contents2009-11-22T19:10:56Z<p>Judy Voet: </p>
<hr />
<div>{{#tree:id=siteTree|openlevels=1|root='''Chemistry of Life'''|close=top|open=top|<br />
<br />
* INTRODUCTION<br />
** Introduction to the Chemistry of Life<br />
<br />
** WATER<br />
<br />
*** [[Water_in_macromolecular_models|Water in Macromolecular Models]]<br />
** KEY CHEMICAL CONCEPTS FOR STRUCTURAL BIOLOGY<br />
<br />
*** [[Cation-pi interactions]]<br />
*** [[Hydrogen bond]]<br />
*** [[Hydrogen_in_macromolecular_models|Hydrogen in Macromolecular Models]]<br />
*** [[Isoelectric_point]]<br />
<br />
* BIOMOLECULES<br />
**[[About_Macromolecular_Structure]]<br />
<br />
** NUCLEOTIDES, NUCLEIC ACIDS AND GENETIC INFORMATION<br />
*** DNA<br />
**** [[DNA]]<br />
**** B-DNA [[1bna]]<br />
**** [[Z-DNA]]<br />
<br />
** AMINO ACIDS<br />
*** [[Amino_Acids]]<br />
*** [[Selenocysteine]]<br />
** PROTEINS: PRIMARY STRUCTURE<br />
*** [[Conservation,_Evolutionary]]<br />
*** [[Isoelectric_point]]<br />
<br />
<br />
** PROTEINS: THREE-DIMENSIONAL STRUCTURE<br />
*** [[S347/The four tiers of protein structure]]<br />
*** [[Secondary_structure]]<br />
*** Protein structural motifs<br />
**** [[User:James_D_Watson/Structural_Templates]]<br />
*** FIBROUS PROTEINS<br />
**** [[Coiled_coil]]<br />
**** [[Collagen]]<br />
*** Protein Misfolding<br />
**** [[Prion_protein]]<br />
**** [[A_Physical_Model_of_the_Structure_of_GNNQQNY_from_Yeast_Prion_Sup35]]<br />
<br />
** COLORED PROTEINS & THOSE THAT CHANGE COLOR<br />
*** [[Green Fluorescent Protein]]; [[GFP_(Hebrew)]]<br />
*** [[Dronpa]]<br />
*** [[Factor IX]]<br />
<br />
** DNA BINDING PROTEINS<br />
*** [[Helix-turn-helix motif]]<br />
*** [[DNA-binding protein VirE2 from Agrobacterium tumefaciens complexed with chaperone VirE1]]<br />
<br />
** PROTEIN FUNCTION: Myoglobin and Hemoglobin, Muscle Contraction, and Antibodies<br />
*** Antibodies<br />
**** [[IgA]]<br />
**** [[Epitopes]]<br />
**** [[Major_Histocompatibility_Complex_Class_I]]<br />
*** [[Calmodulin_in_motion]]<br />
*** [[Myoglobin]]<br />
*** [[Hemoglobin]]; [[Hemoglobin_(Hebrew)]]<br />
*** [[Kinesin-5]]<br />
<br />
** LIPIDS AND BIOLOGICAL MEMBRANES<br />
<br />
** MEMBRANE TRANSPORT PROTEINS<br />
*** [[A Physical Model of the β2-Adrenergic Receptor]]<br />
*** [[Lactose_Permease]]<br />
*** [[Proton_Channels]]<br />
*** [[Ion_channels]]<br />
*** [[Mechanosensitive_channels:_opening_and_closing]]<br />
***[[Enzyme_I_of_the_Phosphoenolpyruvate:Sugar_Phosphotransferase_System]]<br />
<br />
** PRIONS AND INTRINSICALLY DISORDERED PROTEINS<br />
*** [[Prion_protein]]<br />
*** [[Doppel]]<br />
*** [[Intrinsically Disordered Protein]]<br />
<br />
** TOXINS<br />
*** [[Insecticidal delta-endotoxin Cyt2Ba from Bacillus thuringiensis]]<br />
<br />
** MITOSIS, MEOSIS, AND CARGO TRANSPORT PROTEINS<br />
*** [[Kinesin-5]]<br />
<br />
** VIRUSES<br />
*** [[User:Wayne_Decatur/Suppression_of_RNA_Silencing_by_Viruses]]<br />
*** Filamentous bacteriphage<br />
**** [[G3p]] - minor coat protein found on the surface of filamentous bacteriophage<br />
*** HIV<br />
**** [[HIV-1 protease]]<br />
**** [[HIV-1 Gag]]<br />
**** [[HIV-1 Gag Recruitment of Tsg101 and the Viral Budding Process]]<br />
**** [[User:Eric_Martz/Molecular_Playground/HIVDrug]]<br />
*** Herpes Simplex Virus<br />
**** [[Herpes_Simplex_Virus_Thymidine_Kinase]]<br />
*** Influenza<br />
**** [[Influenza_hemagglutinin]]<br />
**** [[Avian_Influenza_Neuraminidase,_Tamiflu_and_Relenza]]<br />
<br />
* ENZYMES<br />
** Enzymatic Catalysis<br />
***[[Hen_Egg-White_%28HEW%29_Lysozyme]]<br />
*** Serine Hydrolases<br />
**** [[Serine_Protease]]<br />
**** [[Trypsin]]<br />
**** [[Alpha-1-antitrypsin]]<br />
**** [[Acetylcholinesterase]] (AChE)<br />
***** [[Acetylcholine]]<br />
***** [[Flexibility_of_aromatic_residues_in_acetylcholinesterase]]<br />
***** [[AChE inhibitors and substrates]]<br />
****** [[1eve]] AChE-Aricept complex; [[1eve (Chinese)]]; [[1eve (Russian)]]; [[1eve (Spanish)]]; [[1eve (Turkish)]]<br />
****** [[AChE_bivalent_inhibitors]]<br />
*** Cysteine Proteases<br />
**** [[Tobacco_Etch_Virus_(TEV)_Protease]]<br />
**** [[Streptomyces_griseus_Aminopeptidase_(SGAP)]]; [[Aminopeptidase]]<br />
*** Acid Proteases<br />
**** [[HIV-1 protease]]<br />
**** [[Pepsin]]<br />
*** Metaloproteases<br />
**** [[Metalloproteases]]<br />
**** [[Matrix_metalloproteinases]]<br />
*** Oxidoreductases<br />
**** [[NADH quinone oxidoreductase ]]<br />
*** [[Triose_Phosphate_Isomerase]]<br />
*** [[Aconitase]]<br />
*** [[Enzyme I of the Phosphoenolpyruvate:Sugar Phosphotransferase System]]<br />
*** [[Pyruvate_phosphate_dikinase]]<br />
***[[Phosphofructokinase_%28PFK%29]]<br />
<br />
** Enzyme Kinetics, Inhibition, and Control<br />
***[[Avian_Influenza_Neuraminidase,_Tamiflu_and_Relenza]]<br />
<br />
** BIOCHEMICAL SIGNALING<br />
*** [[Nitric_oxide_synthase]]<br />
*** [[Recoverin,_a_calcium-activated_myristoyl_switch]]<br />
*** [[Bcl-2]]<br />
*** [[C-Myc]]<br />
<br />
* METABOLISM<br />
** Introduction to Metabolism<br />
<br />
** Glucose Catabolism<br />
*** [[Triose_Phosphate_Isomerase]]<br />
***[[Phosphofructokinase_%28PFK%29]]<br />
<br />
<br />
** Glycogen Metabolism and Gluconeogenesis<br />
*** [[Phosphoglucose_isomerase]]<br />
*** [[Calmodulin_in_motion]]<br />
*** [[Biotin_Protein_Ligase]]<br />
<br />
** Citric Acid Cycle<br />
*** [[Aconitase]]<br />
<br />
** Electron Transport and Oxidative Phosphorylation<br />
*** [[NADH quinone oxidoreductase ]]<br />
<br />
** Photosynthesis<br />
*** [[Photosystem_II]]<br />
*** [[Ribulose-1,5-bisphosphate_carboxylase/oxygenase]]<br />
*** [[PrrA_in_Rhodobacter_sphaeroides]]<br />
*** [[Pyruvate_phosphate_dikinase]]<br />
<br />
** Lipid Metabolism<br />
*** [[Acid-beta-glucosidase]]<br />
** Amino Acid Metabolism<br />
*** [[Aromatic_amino_acid_hydroxylases]]<br />
*** [[Phenylalanine_hydroxylase]]<br />
*** [[Tyrosine_hydroxylase]]<br />
*** [[ATP_Phosphoribosyl_Transferase]]<br />
*** [[Isochorismate_pyruvate_lyase]]<br />
** Mammalian Fuel Metabolism: Integration and Regulation<br />
** Nucleotide Metabolism<br />
*** [[Dihydrofolate_reductase]]<br />
<br />
* GENE EXPRESSION AND REPLICATION <br />
<br />
<br />
** Nucleic Acid Structure<br />
*** [[DNA]]<br />
**** B-DNA [[1bna]]<br />
**** [[Z-DNA]]<br />
*** [[Nucleosomes]]<br />
** DNA Replication, Repair, and Recombination<br />
*** [[DNA Replication,Transcription and Translation]]<br />
*** [[PcrA_helicase]]<br />
*** [[Fpg_Nei_Protein_Superfamily]] - DNA Repair and Base Excision DNA Repair<br />
*** [[Human RecQ-Like protein 1]] - RecQ family of DNA helicases are conserved in from bacteria to man <br />
*** [[Structure_of_E._coli_DnaC_helicase_loader]]<br />
***[[1x9n#Crystal_Structure_of_Human_DNA_Ligase_I_bound_to_5.27-adenylated.2C_nicked_DNA]]<br />
*** [[Rop_protein]]<br />
<br />
** Transcription and RNA Processing<br />
*** [[C-Myc]]<br />
*** [[Lac_repressor]]<br />
*** [[TATA-Binding_Protein]]<br />
*** [[RSP1275]]<br />
*** [[Transcription_Termination_Factor_Rho]]<br />
<br />
** Protein Synthesis<br />
*** [[Ribosome]]<br />
*** [[SelB_Recognition]]<br />
** Regulation of Gene Expression<br />
*** [[Lac_repressor]]<br />
*** [[Irr]]<br />
*** [[P53]]<br />
*** [[Tangible_Models_of_Cdc42_Interacting_With_Intersectin]]<br />
<br />
* EVOLUTION<br />
** [[Conservation, Evolutionary]]<br />
<br />
** [[Extremophiles]]<br />
<br />
* IMMUNE SYSTEM<br />
** Antibodies<br />
*** [[IgA]]<br />
*** [[Epitopes]]<br />
** [[Major_Histocompatibility_Complex_Class_I]]<br />
<br />
<br />
<br />
* METHODS OF STRUCTURE DETERMINATION <br />
** X-ray<br />
*** [[X-ray_crystallography]]<br />
**** [[Asymmetric_Unit]]<br />
**** [[Biological_Unit]]<br />
**** [[Electron_density_maps]]<br />
*** SAXS<br />
** NMR<br />
*** [[NMR_Ensembles_of_Models]]<br />
** Electron Microscopy<br />
<br />
* METHODS OF STRUCTURE & SEQUENCE ANALYSIS <br />
** STRUCTURAL ANALYSIS & VISUALIZATION<br />
*** [[Chime]]<br />
*** Jmol<br />
**** [[S347/Visualising_protein_structure|Visualising Protein Structure]] - Introduction to Jmol<br />
**** [[FirstGlance_in_Jmol]]<br />
*** [[User:Wayne Decatur/Teaching Proteopedia|Teaching Proteopedia]]<br />
*** [[User:Wayne Decatur/Generate Unfolded Structures|Generate Unfolded Structures]]<br />
*** Homology Model<br />
**** [[User:Wayne Decatur/Homology Modeling|Homology Modeling]]<br />
**** [[User:Emi Nakayama/TRIM5a Homology Models|TRIM5a Homology Models]] - Differences in models as a function of their templates<br />
*** [[DRuMS]] - set of standard color schemes for macromolecular visualization<br />
<br />
** SEQUENCE ANALYSIS<br />
*** [[User:Wayne Decatur/Sequence analysis tools|Sequence Analysis Tools]]<br />
<br />
}}</div>Judy Voethttps://proteopedia.org/index.php?title=Phosphofructokinase_(PFK)&diff=1020092Phosphofructokinase (PFK)2009-11-22T19:08:15Z<p>Judy Voet: New page: ==Overview== Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bispho...</p>
<hr />
<div>==Overview==<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, PEP and fructose-2,6-bisphosphate (F2,6P). <br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<scene name='Sandbox/Judy_Voet/PFK/4pfk_biol/1'>PFK from B. stearothermophilus</scene> is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_overview/2'>subunits A (light Blue) and D (yellow)</scene> with the active site interfaces in magenta with the substrates in cyan. Two more active sites are at the interface of subunits B (green) and C (pink). A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/2'>A/D interface active site</scene> of subunit D (Yellow) shows that amino acids from both subunits A (light blue) and D (Yellow) contribute to the binding of F6P. Two of the allosteric sites are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_overview/2'>subunits A and B</scene> and two at the interface of subunits C and D. Again the interfaces are magenta with the allosteric ligand in cyan. A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_closeup/4'>A/B allosteric site</scene> of subunit A shows contributions from both subunits to the binding of ADP. The conformational changes in going between the R and T states of PFK are illustrated below.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
== Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1: PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are part of the F6P binding site in the T and R states, srespectively(see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2: Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three residues (2 arg and 1 Lys; not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1: The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the absence of the positive charge of Arg 162 have on the binding of F6P? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2: Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
At one time, the negative charge of Glu 161 was thought to have a negative effect on F6P binding in the T state. This idea has not been supported by site-directed mutagenesis experiments<ref>PMID:10759544</ref>.Several mutant PFKs have been made, including R162A, E161A and R162A/E161A. The R162A mutation caused a 30-fold decrease in F6P binding. The E161A mutation, however, had little effect on the ability of PEP to inhibit F6P binding. <br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/></div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1020091Sandbox/Judy Voet/PFK2009-11-22T19:04:17Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, PEP and fructose-2,6-bisphosphate (F2,6P). <br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<scene name='Sandbox/Judy_Voet/PFK/4pfk_biol/1'>PFK from B. stearothermophilus</scene> is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_overview/2'>subunits A (light Blue) and D (yellow)</scene> with the active site interfaces in magenta with the substrates in cyan. Two more active sites are at the interface of subunits B (green) and C (pink). A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/2'>A/D interface active site</scene> of subunit D (Yellow) shows that amino acids from both subunits A (light blue) and D (Yellow) contribute to the binding of F6P. Two of the allosteric sites are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_overview/2'>subunits A and B</scene> and two at the interface of subunits C and D. Again the interfaces are magenta with the allosteric ligand in cyan. A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_closeup/4'>A/B allosteric site</scene> of subunit A shows contributions from both subunits to the binding of ADP. The conformational changes in going between the R and T states of PFK are illustrated below.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1: PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are part of the F6P binding site in the T and R states, srespectively(see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2: Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three residues (2 arg and 1 Lys; not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1: The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the absence of the positive charge of Arg 162 have on the binding of F6P? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2: Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
At one time, the negative charge of Glu 161 was thought to have a negative effect on F6P binding in the T state. This idea has not been supported by site-directed mutagenesis experiments<ref>PMID:10759544</ref>.Several mutant PFKs have been made, including R162A, E161A and R162A/E161A. The R162A mutation caused a 30-fold decrease in F6P binding. The E161A mutation, however, had little effect on the ability of PEP to inhibit F6P binding. <br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/></div>Judy Voethttps://proteopedia.org/index.php?title=Proteopedia:Table_of_Contents&diff=1020090Proteopedia:Table of Contents2009-11-22T18:50:26Z<p>Judy Voet: </p>
<hr />
<div>{{#tree:id=siteTree|openlevels=1|root='''Chemistry of Life'''|close=top|open=top|<br />
<br />
* INTRODUCTION<br />
** Introduction to the Chemistry of Life<br />
<br />
** WATER<br />
<br />
*** [[Water_in_macromolecular_models|Water in Macromolecular Models]]<br />
** KEY CHEMICAL CONCEPTS FOR STRUCTURAL BIOLOGY<br />
<br />
*** [[Cation-pi interactions]]<br />
*** [[Hydrogen bond]]<br />
*** [[Hydrogen_in_macromolecular_models|Hydrogen in Macromolecular Models]]<br />
*** [[Isoelectric_point]]<br />
<br />
* BIOMOLECULES<br />
**[[About_Macromolecular_Structure]]<br />
<br />
** NUCLEOTIDES, NUCLEIC ACIDS AND GENETIC INFORMATION<br />
*** DNA<br />
**** [[DNA]]<br />
**** B-DNA [[1bna]]<br />
**** [[Z-DNA]]<br />
<br />
** AMINO ACIDS<br />
*** [[Amino_Acids]]<br />
*** [[Selenocysteine]]<br />
** PROTEINS: PRIMARY STRUCTURE<br />
*** [[Conservation,_Evolutionary]]<br />
*** [[Isoelectric_point]]<br />
<br />
<br />
** PROTEINS: THREE-DIMENSIONAL STRUCTURE<br />
*** [[S347/The four tiers of protein structure]]<br />
*** [[Secondary_structure]]<br />
*** Protein structural motifs<br />
**** [[User:James_D_Watson/Structural_Templates]]<br />
*** FIBROUS PROTEINS<br />
**** [[Coiled_coil]]<br />
**** [[Collagen]]<br />
*** Protein Misfolding<br />
**** [[Prion_protein]]<br />
**** [[A_Physical_Model_of_the_Structure_of_GNNQQNY_from_Yeast_Prion_Sup35]]<br />
<br />
** COLORED PROTEINS & THOSE THAT CHANGE COLOR<br />
*** [[Green Fluorescent Protein]]; [[GFP_(Hebrew)]]<br />
*** [[Dronpa]]<br />
*** [[Factor IX]]<br />
<br />
** DNA BINDING PROTEINS<br />
*** [[Helix-turn-helix motif]]<br />
*** [[DNA-binding protein VirE2 from Agrobacterium tumefaciens complexed with chaperone VirE1]]<br />
<br />
** PROTEIN FUNCTION: Myoglobin and Hemoglobin, Muscle Contraction, and Antibodies<br />
*** Antibodies<br />
**** [[IgA]]<br />
**** [[Epitopes]]<br />
**** [[Major_Histocompatibility_Complex_Class_I]]<br />
*** [[Calmodulin_in_motion]]<br />
*** [[Myoglobin]]<br />
*** [[Hemoglobin]]; [[Hemoglobin_(Hebrew)]]<br />
*** [[Kinesin-5]]<br />
<br />
** LIPIDS AND BIOLOGICAL MEMBRANES<br />
<br />
** MEMBRANE TRANSPORT PROTEINS<br />
*** [[A Physical Model of the β2-Adrenergic Receptor]]<br />
*** [[Lactose_Permease]]<br />
*** [[Proton_Channels]]<br />
*** [[Ion_channels]]<br />
*** [[Mechanosensitive_channels:_opening_and_closing]]<br />
***[[Enzyme_I_of_the_Phosphoenolpyruvate:Sugar_Phosphotransferase_System]]<br />
<br />
** PRIONS AND INTRINSICALLY DISORDERED PROTEINS<br />
*** [[Prion_protein]]<br />
*** [[Doppel]]<br />
*** [[Intrinsically Disordered Protein]]<br />
<br />
** TOXINS<br />
*** [[Insecticidal delta-endotoxin Cyt2Ba from Bacillus thuringiensis]]<br />
<br />
** MITOSIS, MEOSIS, AND CARGO TRANSPORT PROTEINS<br />
*** [[Kinesin-5]]<br />
<br />
** VIRUSES<br />
*** [[User:Wayne_Decatur/Suppression_of_RNA_Silencing_by_Viruses]]<br />
*** Filamentous bacteriphage<br />
**** [[G3p]] - minor coat protein found on the surface of filamentous bacteriophage<br />
*** HIV<br />
**** [[HIV-1 protease]]<br />
**** [[HIV-1 Gag]]<br />
**** [[HIV-1 Gag Recruitment of Tsg101 and the Viral Budding Process]]<br />
**** [[User:Eric_Martz/Molecular_Playground/HIVDrug]]<br />
*** Herpes Simplex Virus<br />
**** [[Herpes_Simplex_Virus_Thymidine_Kinase]]<br />
*** Influenza<br />
**** [[Influenza_hemagglutinin]]<br />
**** [[Avian_Influenza_Neuraminidase,_Tamiflu_and_Relenza]]<br />
<br />
* ENZYMES<br />
** Enzymatic Catalysis<br />
***[[Hen_Egg-White_%28HEW%29_Lysozyme]]<br />
*** Serine Hydrolases<br />
**** [[Serine_Protease]]<br />
**** [[Trypsin]]<br />
**** [[Alpha-1-antitrypsin]]<br />
**** [[Acetylcholinesterase]] (AChE)<br />
***** [[Acetylcholine]]<br />
***** [[Flexibility_of_aromatic_residues_in_acetylcholinesterase]]<br />
***** [[AChE inhibitors and substrates]]<br />
****** [[1eve]] AChE-Aricept complex; [[1eve (Chinese)]]; [[1eve (Russian)]]; [[1eve (Spanish)]]; [[1eve (Turkish)]]<br />
****** [[AChE_bivalent_inhibitors]]<br />
*** Cysteine Proteases<br />
**** [[Tobacco_Etch_Virus_(TEV)_Protease]]<br />
**** [[Streptomyces_griseus_Aminopeptidase_(SGAP)]]; [[Aminopeptidase]]<br />
*** Acid Proteases<br />
**** [[HIV-1 protease]]<br />
**** [[Pepsin]]<br />
*** Metaloproteases<br />
**** [[Metalloproteases]]<br />
**** [[Matrix_metalloproteinases]]<br />
*** Oxidoreductases<br />
**** [[NADH quinone oxidoreductase ]]<br />
*** [[Triose_Phosphate_Isomerase]]<br />
*** [[Aconitase]]<br />
*** [[Enzyme I of the Phosphoenolpyruvate:Sugar Phosphotransferase System]]<br />
*** [[Pyruvate_phosphate_dikinase]]<br />
<br />
** Enzyme Kinetics, Inhibition, and Control<br />
***[[Avian_Influenza_Neuraminidase,_Tamiflu_and_Relenza]]<br />
<br />
** BIOCHEMICAL SIGNALING<br />
*** [[Nitric_oxide_synthase]]<br />
*** [[Recoverin,_a_calcium-activated_myristoyl_switch]]<br />
*** [[Bcl-2]]<br />
*** [[C-Myc]]<br />
<br />
* METABOLISM<br />
** Introduction to Metabolism<br />
<br />
** Glucose Catabolism<br />
*** [[Triose_Phosphate_Isomerase]]<br />
** Glycogen Metabolism and Gluconeogenesis<br />
*** [[Phosphoglucose_isomerase]]<br />
*** [[Calmodulin_in_motion]]<br />
*** [[Biotin_Protein_Ligase]]<br />
<br />
** Citric Acid Cycle<br />
*** [[Aconitase]]<br />
<br />
** Electron Transport and Oxidative Phosphorylation<br />
*** [[NADH quinone oxidoreductase ]]<br />
<br />
** Photosynthesis<br />
*** [[Photosystem_II]]<br />
*** [[Ribulose-1,5-bisphosphate_carboxylase/oxygenase]]<br />
*** [[PrrA_in_Rhodobacter_sphaeroides]]<br />
*** [[Pyruvate_phosphate_dikinase]]<br />
<br />
** Lipid Metabolism<br />
*** [[Acid-beta-glucosidase]]<br />
** Amino Acid Metabolism<br />
*** [[Aromatic_amino_acid_hydroxylases]]<br />
*** [[Phenylalanine_hydroxylase]]<br />
*** [[Tyrosine_hydroxylase]]<br />
*** [[ATP_Phosphoribosyl_Transferase]]<br />
*** [[Isochorismate_pyruvate_lyase]]<br />
** Mammalian Fuel Metabolism: Integration and Regulation<br />
** Nucleotide Metabolism<br />
*** [[Dihydrofolate_reductase]]<br />
<br />
* GENE EXPRESSION AND REPLICATION <br />
<br />
<br />
** Nucleic Acid Structure<br />
*** [[DNA]]<br />
**** B-DNA [[1bna]]<br />
**** [[Z-DNA]]<br />
*** [[Nucleosomes]]<br />
** DNA Replication, Repair, and Recombination<br />
*** [[DNA Replication,Transcription and Translation]]<br />
*** [[PcrA_helicase]]<br />
*** [[Fpg_Nei_Protein_Superfamily]] - DNA Repair and Base Excision DNA Repair<br />
*** [[Human RecQ-Like protein 1]] - RecQ family of DNA helicases are conserved in from bacteria to man <br />
*** [[Structure_of_E._coli_DnaC_helicase_loader]]<br />
***[[1x9n#Crystal_Structure_of_Human_DNA_Ligase_I_bound_to_5.27-adenylated.2C_nicked_DNA]]<br />
*** [[Rop_protein]]<br />
<br />
** Transcription and RNA Processing<br />
*** [[C-Myc]]<br />
*** [[Lac_repressor]]<br />
*** [[TATA-Binding_Protein]]<br />
*** [[RSP1275]]<br />
*** [[Transcription_Termination_Factor_Rho]]<br />
<br />
** Protein Synthesis<br />
*** [[Ribosome]]<br />
*** [[SelB_Recognition]]<br />
** Regulation of Gene Expression<br />
*** [[Lac_repressor]]<br />
*** [[Irr]]<br />
*** [[P53]]<br />
*** [[Tangible_Models_of_Cdc42_Interacting_With_Intersectin]]<br />
<br />
* EVOLUTION<br />
** [[Conservation, Evolutionary]]<br />
<br />
** [[Extremophiles]]<br />
<br />
* IMMUNE SYSTEM<br />
** Antibodies<br />
*** [[IgA]]<br />
*** [[Epitopes]]<br />
** [[Major_Histocompatibility_Complex_Class_I]]<br />
<br />
<br />
<br />
* METHODS OF STRUCTURE DETERMINATION <br />
** X-ray<br />
*** [[X-ray_crystallography]]<br />
**** [[Asymmetric_Unit]]<br />
**** [[Biological_Unit]]<br />
**** [[Electron_density_maps]]<br />
*** SAXS<br />
** NMR<br />
*** [[NMR_Ensembles_of_Models]]<br />
** Electron Microscopy<br />
<br />
* METHODS OF STRUCTURE & SEQUENCE ANALYSIS <br />
** STRUCTURAL ANALYSIS & VISUALIZATION<br />
*** [[Chime]]<br />
*** Jmol<br />
**** [[S347/Visualising_protein_structure|Visualising Protein Structure]] - Introduction to Jmol<br />
**** [[FirstGlance_in_Jmol]]<br />
*** [[User:Wayne Decatur/Teaching Proteopedia|Teaching Proteopedia]]<br />
*** [[User:Wayne Decatur/Generate Unfolded Structures|Generate Unfolded Structures]]<br />
*** Homology Model<br />
**** [[User:Wayne Decatur/Homology Modeling|Homology Modeling]]<br />
**** [[User:Emi Nakayama/TRIM5a Homology Models|TRIM5a Homology Models]] - Differences in models as a function of their templates<br />
*** [[DRuMS]] - set of standard color schemes for macromolecular visualization<br />
<br />
** SEQUENCE ANALYSIS<br />
*** [[User:Wayne Decatur/Sequence analysis tools|Sequence Analysis Tools]]<br />
<br />
}}</div>Judy Voethttps://proteopedia.org/index.php?title=Hen_Egg-White_(HEW)_Lysozyme&diff=1020089Hen Egg-White (HEW) Lysozyme2009-11-22T18:46:15Z<p>Judy Voet: New page: <applet load='aln_1H6M_to_1HEW_2.pdb' size='300' frame='true' align='right' caption=' Hen Egg White (HEW) Lysozyme containing a trisaccharide of N-acetylglucosamine (NAG) bound to the act...</p>
<hr />
<div><applet load='aln_1H6M_to_1HEW_2.pdb' size='300' frame='true' align='right' caption=' Hen Egg White (HEW) Lysozyme containing a trisaccharide of N-acetylglucosamine (NAG) bound to the active site, PDBid 1HEW' scene='User:Judy_Voet/Lysozyme/Lysozyme1/16' /><br />
Lysozyme was the first enzyme whose X-ray structure was determined <ref> PMID 5840126</ref><ref>Phillips, D. C. The hen egg white lysozyme molecule. Proc. Natl Acad. Sci. USA 57, 483-495 (1967)</ref>. This <scene name='User:Judy_Voet/Lysozyme/Lysozyme1/15'>scene </scene> shows Hen Egg White (HEW) lysozyme containing a trisaccharide of N-acetylglucosamine (NAG) bound to a cleft in the enzyme. David Phillips, who determined the structure in 1965, saw that the cleft was large enough to fit three more saccharide units. He therefore built a model extending the trisaccharide to a <br />
<scene name='User:Judy_Voet/Lysozyme/Lysozyme1_hexamer/7'>hexasaccharide</scene> that fits into the cleft, labeling the sugar subsites A-F<ref> coordinates of the model kindly provided by Louise Johnson</ref>. Alternately click on <scene name='User:Judy_Voet/Lysozyme/Lysozyme1/15'>trisaccharide</scene> and <scene name='User:Judy_Voet/Lysozyme/Lysozyme1_hexamer/7'>hexasaccharide</scene> to turn the modeled portion of the hexasaccharide on and off.<br />
The interesting thing about the model was that the only way that the hexasaccharide would fit into the cleft was if the 4th saccharide (in subsite D) was strained into a <scene name='User:Judy_Voet/Lysozyme/Half-chair/2'>half-chair conformation</scene>. This conformation is what would be necessary for the formation of an oxocarbenium ion (oxionium ion). When the model was studied, <scene name='User:Judy_Voet/Lysozyme/Glu_35/1'>Glu 35</scene> was found to be in an ideal location to act as a general acid catalyst, 3.34 Angstroms from the bridging oxygen between the 4th and 5th saccharide units. <scene name='User:Judy_Voet/Lysozyme/Asp_52/2'>Asp 52</scene> appeared to be too far away (2.69 angstroms) in the static lysozyme structure to have formed a covalent bond with C1 of the half-chair model in the D site, and no covalent intermediate had ever been detected, so Phillips proposed that it acted as an electrostatic stabilizer of the oxonium ion (referred to as The Phillips Mechanism).<br />
<br />
{{Clear}}<br />
<br />
<applet load='aln_1H6M_to_1HEW_2.pdb' size='300' frame='true' align='right' caption='NAG-2-deoxy-2-fluoro-glucosyl fluoride (NAG2FGlcF) bound to Glu35Gln HEW Lysozyme PDBid 1H6M' scene='User:Judy_Voet/Lysozyme/1h6m/3'/><br />
Then, in 2001, Stephen Withers published <scene name='User:Judy_Voet/Lysozyme/1h6m/3'>1H6M</scene>,<ref>PMID 11518970</ref> in which Glu 35 he had mutated to Gln to remove the general acid catalyst. The substrate contained NAG-2-fluoro-glucosyl fluoride (NAG2FGlcF). The fluoro group on C-1 does not require acid catalysis to be a good leaving group, and the remaining saccharide, in the absence of the acid necessary to catalyse the second step of the reaction, was demonstrated to form a <scene name='User:Judy_Voet/Lysozyme/Covalent/1'> covalent intermediate</scene>. In this <scene name='User:Judy_Voet/Lysozyme/Superposition/2'>superposition</scene> of the half chair model with 1HEW (greens) and the covalent intermediate in 1H6M (blues), note the relatively small motions of Asp 52 and C1 of the sugar ring in going from the model to the covalent intermediate. to observe the motion from the <scene name='User:Judy_Voet/Lysozyme/Asp52_halfchair/1'>half-chair</scene> to the <scene name='User:Judy_Voet/Lysozyme/Covalent/2'>covalent intermediate</scene> just toggle between the two green links. <br />
===Some Useful External Links===<br />
[http://en.wikipedia.org/wiki/Lysozyme Lysozyme]<br />
<br />
[http://en.wikipedia.org/wiki/Glycoside_hydrolase#Retaining_glycoside_hydrolases Retaining Glycoside Hydrolases]<br />
===References===<br />
<references/></div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1020035Sandbox/Judy Voet/PFK2009-11-22T14:02:48Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, PEP and fructose-2,6-bisphosphate (F2,6P). <br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<scene name='Sandbox/Judy_Voet/PFK/4pfk_biol/1'>PFK from B. stearothermophilus</scene> is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_overview/2'>subunits A (light Blue) and D (yellow)</scene> with the active site interfaces in magenta with the substrates in cyan. Two more active sites are at the interface of subunits B (green) and C (pink). A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/2'>A/D interface active site</scene> of subunit D (Yellow) shows that amino acids from both subunits A (light blue) and D (Yellow) contribute to the binding of F6P. Two of the allosteric sites are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_overview/2'>subunits A and B</scene> and two at the interface of subunits C and D. Again the interfaces are magenta with the allosteric ligand in cyan. A closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_closeup/4'>A/B allosteric site</scene> of subunit A shows contributions from both subunits to the binding of ADP.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1. PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are part of the F6P binding site in the T and R states, srespectively(see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2. Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three residues (2 arg and 1 Lys; not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1, The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the absence of the positive charge of Arg 162 have on the binding of F6P? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2. Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
At one time, the negative charge of Glu 161 was thought to have a negative effect on F6P binding in the T state. This idea has not been supported by site-directed mutagenesis experiments<ref>PMID:10759544</ref>.Several mutant PFKs have been made, including R162A, E161A and R162A/E161A . The R162A mutation caused a 30-fold decrease in F6P binding. The E161A mutation, however, had little effect on the ability of PEP to inhibit F6P binding. <br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/></div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1020030Sandbox/Judy Voet/PFK2009-11-22T13:10:09Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, PEP and fructose-2,6-bisphosphate (F2,6P). <br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<scene name='Sandbox/Judy_Voet/PFK/4pfk_biol/1'>PFK from B. stearothermophilus</scene> is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_overview/1'>subunits A (light Blue) and D (yellow)</scene> with the active site interfaces in magenta with the substrates in cyan. Two more active sites are at the interface of subunits B (green) and C (pink). Two of the allosteric sites are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_overview/2'>subunits A and B</scene> and two at the interface of subunits C and D. Here is a closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/2'>A/D interface active site</scene> of subunit D (Yellow). Note that amino acids from subunit A (light blue) also contribute to the binding of F6P. Also here is a closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_closeup/2'>A/B allosteric site</scene> of subunit B with contributions from both subunits to the binding of ADP.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1. PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are part of the F6P binding site in the T and R states, srespectively(see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2. Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1, The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the absence of the positive charge of Arg 162 have on the binding of F6P? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2. Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
At one time, the negative charge of Glu 161 was thought to have a negative effect on F6P binding in the T state. This idea has not been supported by site-directed mutagenesis experiments<ref>PMID:10759544</ref>.Several mutant PFKs have been made, including R162A, E161A and R162A/E161A . The R162A mutation caused a 30-fold decrease in F6P binding. The E161A mutation, however, had little effect on the ability of PEP to inhibit F6P binding. <br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/></div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1020007Sandbox/Judy Voet/PFK2009-11-22T11:34:53Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, PEP and fructose-2,6-bisphosphate (F2,6P). <br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<scene name='Sandbox/Judy_Voet/PFK/4pfk_biol/1'>PFK from B. stearothermophilus</scene> is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_overview/1'>subunits A (light Blue) and D (yellow)</scene> with the active site interfaces in magenta with the substrates in cyan. Two more active sites are at the interface of subunits B (green) and C (pink). Two of the allosteric sites are located at the interface of subunits A and B and two at the interface of subunits C and D. Here is a closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/2'>A/D interface active site</scene> of subunit D (Yellow). Note that amino acids from subunit A (light blue) also contribute to the binding of F6P. Also here is a closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_closeup/2'>A/B allosteric site</scene> of subunit B with contributions from both subunits to the binding of ADP.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1. PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are part of the F6P binding site in the T and R states, srespectively(see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2. Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1, The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the absence of the positive charge of Arg 162 have on the binding of F6P? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2. Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
At one time, the negative charge of Glu 161 was thought to have a negative effect on F6P binding in the T state. This idea has not been supported by site-directed mutagenesis experiments<ref>PMID:10759544</ref>.Several mutant PFKs have been made, including R162A, E161A and R162A/E161A . The R162A mutation caused a 30-fold decrease in F6P binding. The E161A mutation, however, had little effect on the ability of PEP to inhibit F6P binding. <br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/></div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1020002Sandbox/Judy Voet/PFK2009-11-22T11:06:06Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, PEP and fructose-2,6-bisphosphate (F2,6P). <br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<scene name='Sandbox/Judy_Voet/PFK/4pfk_biol/1'>PFK from B. stearothermophilus</scene> is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_overview/1'>subunits A (light Blue) and D (yellow)</scene> with the active site interfaces in magenta with the substrates in chan. Two more active sites are at the interface of subunits B (green) and C (pink). Two of the allosteric sites are located at the interface of subunits A and B and two at the interface of subunits C and D. Here is a closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/1'>A/D interface active site</scene> of subunit D (Yellow). Note that amino acids from subunit A (light blue) also contribute to the binding of F6P. Also here is a closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_closeup/2'>A/B allosteric site</scene> of subunit B with contributions from both subunits to the binding of ADP.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1. PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are part of the F6P binding site in the T and R states, srespectively(see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2. Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1, The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the absence of the positive charge of Arg 162 have on the binding of F6P? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2. Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
At one time, the negative charge of Glu 161 was thought to have a negative effect on F6P binding in the T state. This idea has not been supported by site-directed mutagenesis experiments<ref>PMID:10759544</ref>.Several mutant PFKs have been made, including R162A, E161A and R162A/E161A . The R162A mutation caused a 30-fold decrease in F6P binding. The E161A mutation, however, had little effect on the ability of PEP to inhibit F6P binding. <br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/></div>Judy Voethttps://proteopedia.org/index.php?title=Aricept_Complexed_with_Acetylcholinesterase_(Arabic)&diff=1020001Aricept Complexed with Acetylcholinesterase (Arabic)2009-11-22T10:37:35Z<p>Judy Voet: New page: 250px<br /> {{STRUCTURE_1eve| PDB=1eve | SCENE=Main_Page/E2020_in_ache_spinning/1 }} '''3D structure of anti-Alzheimer's drug, Aric...</p>
<hr />
<div>[[Image:E2020_interactins_in_AChE_gorge.jpg|left|250px]]<br /><br />
<br />
{{STRUCTURE_1eve| PDB=1eve | SCENE=Main_Page/E2020_in_ache_spinning/1 }}<br />
<br />
'''3D structure of anti-Alzheimer's drug, Aricept, complexed with acetylcholinesterase'''<br />
(see also [[AChE bivalent inhibitors (Part II)]])<br />
==Background==<br />
Several cholinesterase inhibitors are either being utilized for symptomatic treatment of Alzheimer's disease or are in advanced clinical trials. '''E2020''', marketed as '''Aricept''', is a member of a large family of N-benzylpiperidine-based [[acetylcholinesterase]] (AChE) inhibitors, developed, synthesized and evaluated by the Eisai Company in Japan. These inhibitors were designed on the basis of QSAR studies prior to elucidation of the 3D structure of ''Torpedo californica'' AChE (''Tc''AChE) ([[1ea5]]). It significantly enhances performance in animal models of cholinergic hypofunction and has a high affinity for AChE, binding to both electric eel and mouse AChE in the nanomolar range.<br />
<br />
==Results==<br />
The X-ray structure of the E2020-''Tc''AChE complex shows that E2020 has a <scene name='1eve/E2020_close_up_with_84_279/10'>unique orientation</scene> along the active-site gorge, extending from the anionic subsite (<scene name='1eve/E2020_close_up_with_84lbld/5'>W84</scene>) of the active site, at the bottom, to the peripheral anionic site (<scene name='1eve/E2020_close_up_with_84_279lbld/4'>near W279</scene>), at the top, via aromatic stacking interactions with conserved aromatic acid residues. E2020 does not, however, interact directly with either the catalytic triad or the 'oxyanion hole' but only <scene name='1eve/E20_interactionshown/7'>indirectly via solvent molecules</scene>.<br />
<br />
==Conclusions==<br />
The X-ray structure shows, a posteriori, that the design of E2020 took advantage of several important features of the active-site gorge of AChE, to produce a drug with both high affinity for AChE and a high degree of selectivity for AChE versus butyrylcholinesterase (BChE). It also delineates voids within the gorge that are not occupied by E2020 and could provide sites for potential modification of E2020 to produce drugs with improved pharmacological profiles.<br />
<br />
==About this Structure==<br />
1EVE is a [http://en.wikipedia.org/wiki/Protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Torpedo_californica Torpedo californica] with NAG and E20 as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [[Acetylcholinesterase]], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.1.7 3.1.1.7]. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1EVE OCA].<br />
<br />
==Reference==<br />
Structure of acetylcholinesterase complexed with E2020 (Aricept): implications for the design of new anti-Alzheimer drugs., Kryger G, Silman I, Sussman JL, Structure. 1999 Mar 15;7(3):297-307. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=10368299 10368299]<br />
<br />
See [[1eve (Chinese)]], [[1eve (French)]], [[1eve (Russian)]], [[1eve (Spanish)]], & [[1eve (Turkish)]].<br />
[[Category: Acetylcholinesterase]]<br />
[[Category: Single protein]]<br />
[[Category: Torpedo californica]]<br />
[[Category: Kryger, G.]]<br />
[[Category: Silman, I.]]<br />
[[Category: Sussman, J.L.]]<br />
[[Category: E20]]<br />
[[Category: NAG]]<br />
[[Category: alpha/beta hydrolase]]<br />
[[Category: alzheimer's disease]]<br />
[[Category: catalytic triad]]<br />
[[Category: drug]]<br />
[[Category: glycosylated protein]]<br />
[[Category: neurotransmitter cleavage]]<br />
[[Category: serine hydrolase]]<br />
<br />
''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Thu Nov 8 12:39:55 2007''</div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1019988Sandbox/Judy Voet/PFK2009-11-22T08:20:12Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, PEP and fructose-2,6-bisphosphate (F2,6P). <br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<scene name='Sandbox/Judy_Voet/PFK/4pfk_biol/1'>PFK from B. stearothermophilus</scene> is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of subunits A (light Blue) and D (yellow) and two at the interface of subunits B (green) and C (pink). Two of the allosteric sites are located at the interface of subunits A and B and two at the interface of subunits C and D. Here is a closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/1'>A/D interface active site</scene> of subunit D (Yellow). Note that amino acids from subunit A (light blue) also contribute to the binding of F6P. Also here is a closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_closeup/2'>A/B allosteric site</scene> of subunit B with contributions from both subunits to the binding of ADP.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1. PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are part of the F6P binding site in the T and R states, srespectively(see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2. Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1, The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the absence of the positive charge of Arg 162 have on the binding of F6P? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2. Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
At one time, the negative charge of Glu 161 was thought to have a negative effect on F6P binding in the T state. This idea has not been supported by site-directed mutagenesis experiments<ref>PMID:10759544</ref>.Several mutant PFKs have been made, including R162A, E161A and R162A/E161A . The R162A mutation caused a 30-fold decrease in F6P binding. The E161A mutation, however, had little effect on the ability of PEP to inhibit F6P binding. <br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/></div>Judy Voethttps://proteopedia.org/index.php?title=Proteopedia:Table_of_Contents&diff=1019794Proteopedia:Table of Contents2009-11-19T11:30:04Z<p>Judy Voet: </p>
<hr />
<div>{{#tree:id=siteTree|openlevels=1|root='''Chemistry of Life'''|close=top|open=top|<br />
<br />
* INTRODUCTION<br />
** Introduction to the Chemistry of Life<br />
<br />
** WATER<br />
<br />
*** [[Water_in_macromolecular_models|Water in Macromolecular Models]]<br />
** KEY CHEMICAL CONCEPTS FOR STRUCTURAL BIOLOGY<br />
<br />
*** [[Cation-pi interactions]]<br />
*** [[Hydrogen bond]]<br />
*** [[Hydrogen_in_macromolecular_models|Hydrogen in Macromolecular Models]]<br />
*** [[Isoelectric_point]]<br />
<br />
* BIOMOLECULES<br />
**[[About_Macromolecular_Structure]]<br />
<br />
** NUCLEOTIDES, NUCLEIC ACIDS AND GENETIC INFORMATION<br />
*** DNA<br />
**** [[DNA]]<br />
**** B-DNA [[1bna]]<br />
**** [[Z-DNA]]<br />
<br />
** AMINO ACIDS<br />
*** [[Amino_Acids]]<br />
*** [[Selenocysteine]]<br />
** PROTEINS: PRIMARY STRUCTURE<br />
*** [[Conservation,_Evolutionary]]<br />
*** [[Isoelectric_point]]<br />
<br />
<br />
** PROTEINS: THREE-DIMENSIONAL STRUCTURE<br />
*** [[S347/The four tiers of protein structure]]<br />
*** [[Secondary_structure]]<br />
*** Protein structural motifs<br />
**** [[User:James_D_Watson/Structural_Templates]]<br />
*** FIBROUS PROTEINS<br />
**** [[Coiled_coil]]<br />
**** [[Collagen]]<br />
*** Protein Misfolding<br />
**** [[Prion_protein]]<br />
**** [[A_Physical_Model_of_the_Structure_of_GNNQQNY_from_Yeast_Prion_Sup35]]<br />
<br />
** COLORED PROTEINS & THOSE THAT CHANGE COLOR<br />
*** [[Green Fluorescent Protein]]; [[GFP_(Hebrew)]]<br />
*** [[Dronpa]]<br />
*** [[Factor IX]]<br />
<br />
** DNA BINDING PROTEINS<br />
*** [[Helix-turn-helix motif]]<br />
*** [[DNA-binding protein VirE2 from Agrobacterium tumefaciens complexed with chaperone VirE1]]<br />
<br />
** PROTEIN FUNCTION: Myoglobin and Hemoglobin, Muscle Contraction, and Antibodies<br />
*** Antibodies<br />
**** [[IgA]]<br />
**** [[Epitopes]]<br />
**** [[Major_Histocompatibility_Complex_Class_I]]<br />
*** [[Calmodulin_in_motion]]<br />
*** [[Myoglobin]]<br />
*** [[Hemoglobin]]; [[Hemoglobin_(Hebrew)]]<br />
*** [[Kinesin-5]]<br />
<br />
** LIPIDS AND BIOLOGICAL MEMBRANES<br />
<br />
** MEMBRANE TRANSPORT PROTEINS<br />
*** [[A Physical Model of the β2-Adrenergic Receptor]]<br />
*** [[Lactose_Permease]]<br />
*** [[Proton_Channels]]<br />
*** [[Ion_channels]]<br />
*** [[Mechanosensitive_channels:_opening_and_closing]]<br />
***[[Enzyme_I_of_the_Phosphoenolpyruvate:Sugar_Phosphotransferase_System]]<br />
<br />
** PRIONS AND INTRINSICALLY DISORDERED PROTEINS<br />
*** [[Prion_protein]]<br />
*** [[Doppel]]<br />
*** [[Intrinsically Disordered Protein]]<br />
<br />
** TOXINS<br />
*** [[Insecticidal delta-endotoxin Cyt2Ba from Bacillus thuringiensis]]<br />
<br />
** MITOSIS, MEOSIS, AND CARGO TRANSPORT PROTEINS<br />
*** [[Kinesin-5]]<br />
<br />
** VIRUSES<br />
*** [[User:Wayne_Decatur/Suppression_of_RNA_Silencing_by_Viruses]]<br />
*** Filamentous bacteriphage<br />
**** [[G3p]] - minor coat protein found on the surface of filamentous bacteriophage<br />
*** HIV<br />
**** [[HIV-1 protease]]<br />
**** [[HIV-1 Gag]]<br />
**** [[HIV-1 Gag Recruitment of Tsg101 and the Viral Budding Process]]<br />
**** [[User:Eric_Martz/Molecular_Playground/HIVDrug]]<br />
*** Herpes Simplex Virus<br />
**** [[Herpes_Simplex_Virus_Thymidine_Kinase]]<br />
*** Influenza<br />
**** [[Influenza_hemagglutinin]]<br />
**** [[Avian_Influenza_Neuraminidase,_Tamiflu_and_Relenza]]<br />
<br />
* ENZYMES<br />
** Enzymatic Catalysis<br />
*** [[Aconitase]]<br />
*** [[Enzyme I of the Phosphoenolpyruvate:Sugar Phosphotransferase System]]<br />
*** Serine Hydrolases<br />
**** [[Serine_Protease]]<br />
**** [[Trypsin]]<br />
**** [[Alpha-1-antitrypsin]]<br />
**** [[Acetylcholinesterase]] (AChE)<br />
***** [[Acetylcholine]]<br />
***** [[Flexibility_of_aromatic_residues_in_acetylcholinesterase]]<br />
***** [[AChE inhibitors and substrates]]<br />
****** [[1eve]] AChE-Aricept complex; [[1eve (Chinese)]]; [[1eve (Russian)]]; [[1eve (Spanish)]]; [[1eve (Turkish)]]<br />
****** [[AChE_bivalent_inhibitors]]<br />
*** [[Triose_Phosphate_Isomerase]]<br />
*** [[Pyruvate_phosphate_dikinase]]<br />
*** Cysteine Proteases<br />
**** [[Tobacco_Etch_Virus_(TEV)_Protease]]<br />
**** [[Streptomyces_griseus_Aminopeptidase_(SGAP)]]; [[Aminopeptidase]]<br />
*** Acid Proteases<br />
**** [[HIV-1 protease]]<br />
**** [[Pepsin]]<br />
*** Metaloproteases<br />
**** [[Metalloproteases]]<br />
**** [[Matrix_metalloproteinases]]<br />
*** Oxidoreductases<br />
**** [[NADH quinone oxidoreductase ]]<br />
<br />
** Enzyme Kinetics, Inhibition, and Control<br />
***[[Avian_Influenza_Neuraminidase,_Tamiflu_and_Relenza]]<br />
<br />
** BIOCHEMICAL SIGNALING<br />
*** [[Nitric_oxide_synthase]]<br />
*** [[Recoverin,_a_calcium-activated_myristoyl_switch]]<br />
*** [[Bcl-2]]<br />
*** [[C-Myc]]<br />
<br />
* METABOLISM<br />
** Introduction to Metabolism<br />
<br />
** Glucose Catabolism<br />
*** [[Triose_Phosphate_Isomerase]]<br />
** Glycogen Metabolism and Gluconeogenesis<br />
*** [[Phosphoglucose_isomerase]]<br />
*** [[Calmodulin_in_motion]]<br />
*** [[Biotin_Protein_Ligase]]<br />
<br />
** Citric Acid Cycle<br />
*** [[Aconitase]]<br />
<br />
** Electron Transport and Oxidative Phosphorylation<br />
*** [[NADH quinone oxidoreductase ]]<br />
<br />
** Photosynthesis<br />
*** [[Photosystem_II]]<br />
*** [[Ribulose-1,5-bisphosphate_carboxylase/oxygenase]]<br />
*** [[PrrA_in_Rhodobacter_sphaeroides]]<br />
*** [[Pyruvate_phosphate_dikinase]]<br />
<br />
** Lipid Metabolism<br />
*** [[Acid-beta-glucosidase]]<br />
** Amino Acid Metabolism<br />
*** [[Aromatic_amino_acid_hydroxylases]]<br />
*** [[Phenylalanine_hydroxylase]]<br />
*** [[Tyrosine_hydroxylase]]<br />
*** [[ATP_Phosphoribosyl_Transferase]]<br />
*** [[Isochorismate_pyruvate_lyase]]<br />
** Mammalian Fuel Metabolism: Integration and Regulation<br />
** Nucleotide Metabolism<br />
*** [[Dihydrofolate_reductase]]<br />
<br />
* GENE EXPRESSION AND REPLICATION <br />
<br />
<br />
** Nucleic Acid Structure<br />
*** [[DNA]]<br />
**** B-DNA [[1bna]]<br />
**** [[Z-DNA]]<br />
*** [[Nucleosomes]]<br />
** DNA Replication, Repair, and Recombination<br />
*** [[DNA Replication,Transcription and Translation]]<br />
*** [[PcrA_helicase]]<br />
*** [[Fpg_Nei_Protein_Superfamily]] - DNA Repair and Base Excision DNA Repair<br />
*** [[Human RecQ-Like protein 1]] - RecQ family of DNA helicases are conserved in from bacteria to man <br />
*** [[Structure_of_E._coli_DnaC_helicase_loader]]<br />
***[[1x9n#Crystal_Structure_of_Human_DNA_Ligase_I_bound_to_5.27-adenylated.2C_nicked_DNA]]<br />
*** [[Rop_protein]]<br />
<br />
** Transcription and RNA Processing<br />
*** [[C-Myc]]<br />
*** [[Lac_repressor]]<br />
*** [[TATA-Binding_Protein]]<br />
*** [[RSP1275]]<br />
*** [[Transcription_Termination_Factor_Rho]]<br />
<br />
** Protein Synthesis<br />
*** [[Ribosome]]<br />
*** [[SelB_Recognition]]<br />
** Regulation of Gene Expression<br />
*** [[Lac_repressor]]<br />
*** [[Irr]]<br />
*** [[P53]]<br />
*** [[Tangible_Models_of_Cdc42_Interacting_With_Intersectin]]<br />
<br />
* EVOLUTION<br />
** [[Conservation, Evolutionary]]<br />
<br />
** [[Extremophiles]]<br />
<br />
* IMMUNE SYSTEM<br />
** Antibodies<br />
*** [[IgA]]<br />
*** [[Epitopes]]<br />
** [[Major_Histocompatibility_Complex_Class_I]]<br />
<br />
<br />
<br />
* METHODS OF STRUCTURE DETERMINATION <br />
** X-ray<br />
*** [[X-ray_crystallography]]<br />
**** [[Asymmetric_Unit]]<br />
**** [[Biological_Unit]]<br />
**** [[Electron_density_maps]]<br />
*** SAXS<br />
** NMR<br />
*** [[NMR_Ensembles_of_Models]]<br />
** Electron Microscopy<br />
<br />
* METHODS OF STRUCTURE & SEQUENCE ANALYSIS <br />
** STRUCTURAL ANALYSIS & VISUALIZATION<br />
*** [[Chime]]<br />
*** Jmol<br />
**** [[S347/Visualising_protein_structure|Visualising Protein Structure]] - Introduction to Jmol<br />
**** [[FirstGlance_in_Jmol]]<br />
*** [[User:Wayne Decatur/Teaching Proteopedia|Teaching Proteopedia]]<br />
*** [[User:Wayne Decatur/Generate Unfolded Structures|Generate Unfolded Structures]]<br />
*** Homology Model<br />
**** [[User:Wayne Decatur/Homology Modeling|Homology Modeling]]<br />
**** [[User:Emi Nakayama/TRIM5a Homology Models|TRIM5a Homology Models]] - Differences in models as a function of their templates<br />
*** [[DRuMS]] - set of standard color schemes for macromolecular visualization<br />
<br />
** SEQUENCE ANALYSIS<br />
*** [[User:Wayne Decatur/Sequence analysis tools|Sequence Analysis Tools]]<br />
<br />
}}</div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1017407Sandbox/Judy Voet/PFK2009-11-18T14:47:04Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P). <br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of subunits A (light Blue) and D (yellow) and two at the interface of subunits B (green) and C (pink). Two of the allosteric sites are located at the interface of subunits A and B and two at the interface of subunits C and D. Here is a closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/1'>A/D interface active site</scene> of subunit D (Yellow). Note that amino acids from subunit A (light blue) also contribute to the binding of F6P. Also here is a closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_closeup/2'>A/B allosteric site</scene> of subunit B with contributions from both subunits to the binding of ADP.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1. PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are important participants in PFK's allosterically facilitated conformational change (see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2. Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1, The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the presence of the carboxylate group of Glu 161 have on the phosphate group of F6P were it to bind in the active site? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2. Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
Three mutant PFKs have been made, R162A, E161A and R162A/E161A <ref>PMID:10759544</ref>. The R162A mutation caused a 30-fold decrease in F6P binding. Surprisingly, the E161A mutation had little effect on the ability of PEP to inhibit F6P binding. A more complicated analysis of the allosteric effects of PFK have therefore been proposed.<ref>PMID: 14717614</ref><br />
<br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/></div>Judy Voethttps://proteopedia.org/index.php?title=File:PFK_R_T.pdb&diff=1017369File:PFK R T.pdb2009-11-18T14:34:14Z<p>Judy Voet: T and T forms of PFK</p>
<hr />
<div>== Summary ==<br />
T and T forms of PFK<br />
== Licensing ==<br />
{{subst:Non-commercial from license selector}}</div>Judy Voethttps://proteopedia.org/index.php?title=File:6PFK_biol_E_H.pdb&diff=1017368File:6PFK biol E H.pdb2009-11-18T14:31:41Z<p>Judy Voet: R and T PFK superimposed</p>
<hr />
<div>== Summary ==<br />
R and T PFK superimposed<br />
== Licensing ==<br />
{{subst:Non-commercial from license selector}}</div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1017367Sandbox/Judy Voet/PFK2009-11-18T13:47:30Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P). <br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of subunits A (light Blue) and D (yellow) and two at the interface of subunits B (green) and C (pink). Two of the allosteric sites are located at the interface of subunits A and B and two at the interface of subunits C and D. Here is a closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/1'>A/D interface active site</scene> of subunit D (Yellow). Note that amino acids from subunit A (light blue) also contribute to the binding of F6P. Also here is a closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_closeup/1'>A/B allosteric site</scene> of subunit B with contributions from both subunits to the binding of ADP.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1. PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are important participants in PFK's allosterically facilitated conformational change (see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2. Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1, The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the presence of the carboxylate group of Glu 161 have on the phosphate group of F6P were it to bind in the active site? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2. Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
Three mutant PFKs have been made, R162A, E161A and R162A/E161A <ref>PMID:10759544</ref>. The R162A mutation caused a 30-fold decrease in F6P binding. Surprisingly, the E161A mutation had little effect on the ability of PEP to inhibit F6P binding. A more complicated analysis of the allosteric effects of PFK have therefore been proposed.<ref>PMID: 14717614</ref><br />
<br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/></div>Judy Voethttps://proteopedia.org/index.php?title=File:6PFK_biol.pdb&diff=1017366File:6PFK biol.pdb2009-11-18T13:41:39Z<p>Judy Voet: 6PFK T state</p>
<hr />
<div>== Summary ==<br />
6PFK T state<br />
== Licensing ==<br />
{{subst:Non-commercial from license selector}}</div>Judy Voethttps://proteopedia.org/index.php?title=File:4pfk1_biol.pdb&diff=1017365File:4pfk1 biol.pdb2009-11-18T13:41:03Z<p>Judy Voet: new R state PFK</p>
<hr />
<div>== Summary ==<br />
new R state PFK<br />
== Licensing ==<br />
{{subst:Non-commercial from license selector}}</div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1017364Sandbox/Judy Voet/PFK2009-11-18T12:24:29Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P). <br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of subunits A (light Blue) and D (yellow) and two at the interface of subunits B (green) and C (pink). Two of the allosteric sites are located at the interface of subunits A and B and two at the interface of subunits C and D. Here is a closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/1'>A/D interface active site</scene> of subunit D (Yellow). Note that amino acids from subunit A (light blue) also contribute to the binding of F6P. Also here is a closeup of the <scene name='Sandbox/Judy_Voet/PFK/Pfk_ab_closeup/1'>A/B allosteric site</scene>of subunit B with contributions from both subunits to the binding of ADP.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1. PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are important participants in PFK's allosterically facilitated conformational change (see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2. Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1, The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the presence of the carboxylate group of Glu 161 have on the phosphate group of F6P were it to bind in the active site? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2. Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
Three mutant PFKs have been made, R162A, E161A and R162A/E161A <ref>PMID:10759544</ref>. The R162A mutation caused a 30-fold decrease in F6P binding. Surprisingly, the E161A mutation had little effect on the ability of PEP to inhibit F6P binding. A more complicated analysis of the allosteric effects of PFK have therefore been proposed.<ref>PMID: 14717614</ref><br />
<br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/></div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1017357Sandbox/Judy Voet/PFK2009-11-18T11:36:32Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P). <br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of subunits A (light Blue) and D (yellow) and two at the interface of subunits B (green) and C (pink). Two of the allosteric sites are located at the interface of subunits A and B and two at the interface of subunits C and D. Here is a <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/1'>Closeup</scene> of the active site on subunit D (Yellow). Note that amino acids from subunit A (light blue) also contribute to the binding of F6P.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1. PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are important participants in PFK's allosterically facilitated conformational change (see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2. Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1, The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the presence of the carboxylate group of Glu 161 have on the phosphate group of F6P were it to bind in the active site? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2. Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
Three mutant PFKs have been made, R162A, E161A and R162A/E161A <ref>PMID:10759544</ref>. The R162A mutation caused a 30-fold decrease in F6P binding. Surprisingly, the E161A mutation had little effect on the ability of PEP to inhibit F6P binding. A more complicated analysis of the allosteric effects of PFK have therefore been proposed.<ref>PMID: 14717614</ref><br />
<br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/></div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1017355Sandbox/Judy Voet/PFK2009-11-18T11:35:33Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P). <br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of subunits A (light Blue) and D (yellow) and two at the interface of subunits B (green) and C (pink). Two of the allosteric sites are located at the interface of subunits A and B and two at the interface of subunits C and D. Here is a <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/1'>Closeup</scene> of the active site on subunit D (Yellow). Note that amino acids from subunit A (light blue) also contribute to the binding of F6P.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1. PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are important participants in PFK's allosterically facilitated conformational change (see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2. Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1, The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the presence of the carboxylate group of Glu 161 have on the phosphate group of F6P were it to bind in the active site? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2. Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
Three mutant PFKs have been made, R162A, E161A and R162A/E161A <ref>PMID:10759544</ref>. The R162A mutation caused a 30-fold decrease in F6P binding. Surprisingly, the E161A mutation had little effect on the ability of PEP to inhibit F6P binding. A more complicated analysis of the allosteric effects of PFK have therefore been proposed.<ref>PMID: 14717614</ref><br />
<br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/></div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1017354Sandbox/Judy Voet/PFK2009-11-18T11:32:38Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P). <br />
PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
Two of the active sites of the enzyme are located at the interface of subunits A (light Blue) and D (yellow) and two at the interface of subunits B (green) and C (pink). Two of the allosteric sites are located at the interface of subunits A and B and two at the interface of subunits C and D. Here is a <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/1'>Closeup</scene> of the active site on subunit D (Yellow). Note that amino acids from subunit A (light blue) also contribute to the binding of F6P.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1. PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are important participants in PFK's allosterically facilitated conformational change (see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2. Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1, The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the presence of the carboxylate group of Glu 161 have on the phosphate group of F6P were it to bind in the active site? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2. Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
Three mutant PFKs have been made, R162A, E161A and R162A/E161A <ref>PMID:10759544</ref>. The R162A mutation caused a 30-fold decrease in F6P binding. Surprisingly, the E161A mutation had little effect on the ability of PEP to inhibit F6P binding. A more complicated analysis of the allosteric effects of PFK have therefore been proposed.<ref>PMID: 14717614</ref><br />
<br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/></div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1017339Sandbox/Judy Voet/PFK2009-11-18T10:57:14Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P). <br />
PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.<ref>PMID:2136935</ref> The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme. <br />
The Active sites of the enzyme are located at the interface of subunits A (yellow) and D (light blue) and at the interface of subunits B and C. The allosteric sites are located at the interface of subunits A and B and at the interface of subunits C and D. Here is a <scene name='Sandbox/Judy_Voet/PFK/Pfk_ad_closeup/1'>Closeup</scene> of the active site on subunit A (yellow). Note that amino acids from subunit D (light blue) also contribute to the binding of F6P.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1. PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are important participants in PFK's allosterically facilitated conformational change (see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2. Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1, The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the presence of the carboxylate group of Glu 161 have on the phosphate group of F6P were it to bind in the active site? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2. Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
Three mutant PFKs have been made, R162A, E161A and R162A/E161A <ref>PMID:10759544</ref>. The R162A mutation caused a 30-fold decrease in F6P binding. Surprisingly, the E161A mutation had little effect on the ability of PEP to inhibit F6P binding. A more complicated analysis of the allosteric effects of PFK have therefore been proposed.<ref>PMID: 14717614</ref><br />
<br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/></div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1017239Sandbox/Judy Voet/PFK2009-11-17T16:07:46Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P). <br />
The symmetry model of allosterism requires that an oligomeric (multisubunit) protein maintain its molecular symmetry in undergoing a transition from its high-activity R state to its low-activity T state. Hence, this transition must be concerted, with no intermediate states. The X-ray <ref>PMID:2136935</ref>and enzymological evidence indicates that PFK, a tetramer of identical subunits, is an allosteric enzyme that follows the symmetry model. Hence, the binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1. PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are important participants in PFK's allosterically facilitated conformational change (see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2. Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1, The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the presence of the carboxylate group of Glu 161 have on the phosphate group of F6P were it to bind in the active site? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2. Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
Three mutant PFKs have been made, R162A, E161A and R162A/E161A <ref>PMID:10759544</ref>. The R162A mutation caused a 30-fold decrease in F6P binding. Surprisingly, the E161A mutation had little effect on the ability of PEP to inhibit F6P binding. A more complicated analysis of the allosteric effects of PFK have therefore been proposed.<ref>PMID: 14717614</ref><br />
<br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/></div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1017222Sandbox/Judy Voet/PFK2009-11-17T15:04:21Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P). <br />
The symmetry model of allosterism requires that an oligomeric (multisubunit) protein maintain its molecular symmetry in undergoing a transition from its high-activity R state to its low-activity T state. Hence, this transition must be concerted, with no intermediate states. The X-ray <ref>PMID:2136935</ref>and enzymological evidence indicates that PFK, a tetramer of identical subunits, is an allosteric enzyme that follows the symmetry model. Hence, the binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1. PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are important participants in PFK's allosterically facilitated conformational change (see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2. Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1, The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the presence of the carboxylate group of Glu 161 have on the phosphate group of F6P were it to bind in the active site? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2. Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
Three mutant PFKs have been made, R162A, E161A and R162A/E161A <ref>PMID:10759544</ref>. The R162A mutation caused a 30-fold decrease in F6P binding. Surprisingly, the E161A mutation had little effect on the ability of PEP to inhibit F6P binding. A more complicated analysis of the allosteric effects of PFK have therefore been proposed.<br />
<br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<references/><br />
<ref group="xtra">PMID:2136935</ref><references group="xtra"/><br />
<ref group="xtra">PMID:10759544</ref><references group="xtra"/></div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1017220Sandbox/Judy Voet/PFK2009-11-17T14:48:26Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-controlling enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P). <br />
The symmetry model of allosterism requires that an oligomeric (multisubunit) protein maintain its molecular symmetry in undergoing a transition from its high-activity R state to its low-activity T state. Hence, this transition must be concerted, with no intermediate states. The X-ray and enzymological evidence indicates that PFK, a tetramer of identical subunits, is an allosteric enzyme that follows the symmetry model. Hence, the binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
The first view, 1. PFK dimer, shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are important participants in PFK's allosterically facilitated conformational change (see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
The seconbd view, 2. Allo/Act Sites, is a closeup of the upper portion of the first view showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the active site on the adjacent subunit. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in view 1, The Allosteric Site, in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; viewed by clicking on "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the presence of the carboxylate group of Glu 161 have on the phosphate group of F6P were it to bind in the active site? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View 2. Closeup, for a closeup of the F6P-sidechain interactions. Center the molecules by choosing "pickcenter" from the "tools" menu and clicking on athe atom you'd like to be in the center. Slide the "zoom" slider to enlarge the view.<br />
==Site-Directed Mutagenesis==<br />
Three mutant PFKs have been made, R162A, E161A and R162A/E161A <ref>PMID:10759544</ref>. The R162A mutation caused a 30-fold decrease in F6P binding. Surprisingly, the E161A mutation had little effect on the ability of PEP to inhibit F6P binding.<br />
<br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<ref group="xtra">PMID:2136935</ref><references group="xtra"/><br />
<ref group="xtra">PMID:10759544</ref><references group="xtra"/></div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1017195Sandbox/Judy Voet/PFK2009-11-17T08:19:08Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-controlling enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P). <br />
The symmetry model of allosterism requires that an oligomeric (multisubunit) protein maintain its molecular symmetry in undergoing a transition from its high-activity R state to its low-activity T state. Hence, this transition must be concerted, with no intermediate states. The X-ray and enzymological evidence indicates that PFK, a tetramer of identical subunits, is an allosteric enzyme that follows the symmetry model. Hence, the binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. This kinemage shows the two subunits of the tetramer whose interface contains two active sites. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
View1 shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are important participants in PFK's allosterically facilitated conformational change (see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
View2 is a closeup of the upper portion of View1 showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the adjacent active site. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the presence of the carboxylate group of Glu 161 have on the phosphate group of F6P were it to bind in the active site? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View2 for a closeup of the F6P-sidechain interactions.<br />
<br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4pfk; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K. but are now available as 6pfk.<br />
<br />
==References==<br />
<ref group="xtra">PMID:2136935</ref><references group="xtra"/><br />
<ref group="xtra">PMID:10759544</ref><references group="xtra"/></div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox_dvoet/DNA_polymerase&diff=1017115Sandbox dvoet/DNA polymerase2009-11-16T19:57:35Z<p>Judy Voet: /* DNA polymerase I */</p>
<hr />
<div>==DNA polymerase I==<br />
DNA replication is catalyzed by '''DNA polymerase.''' All cells express several different DNA polymerases that variously participate in the several aspects of DNA replication and in the repair of damaged DNA. DNA polymerases catalyze the reaction (DNA)<sub>n residues</sub> + dNTP → (DNA)<sub>n+1 residues</sub> + PP<sub>''i''</sub>, where dNTP is the deoxynucleoside triphosphate whose base is complementary to a base on the strand being copied, the so-called '''template strand.''' In addition, DNA polymerases cannot initiate replication by linking together two dNTPs, but rather, can only link the incoming nucleotide to a terminal 3'-OH group on an existing polynucleotide strand, the so-called '''primer strand,''' thereby forming a 3' → 5' phosphodiester bond between successive deoxynucleotides.<br />
<br />
If DNA polymerase can only add nucleotides to a pre-existing primer strand, how can the primer be synthesized? The answer is that the initial primer is a short RNA strand that is complementary to the a portion of the template strand and which is synthesized by an RNA polymerase known as '''primase.''' This enzyme catalyzes a reaction similar to that catalyzed by DNA polymerase but uses NTPs rather than dNTPs. However primase, as can all RNA polymerases, does not require a primer to initiate polynucleotide synthesis; it can do so by linking together two NTPs in a 3' → 5' linkage.<br />
<br />
The first known DNA polymerase, an ''E. coli'' enzyme now known as '''DNA polymerase I''' or '''Pol I,''' was discovered and characterized in 1957 by Arthur Kornberg (who received the Nobel prize for this work). Pol I has three active sites: <br />
<br />
:1. A DNA polymerase. <br />
<br />
:2. A 3' → 5' exonuclease, that hydrolyzes off mispaired nucleotides at the 3' end of the growing polynucleotide [(DNA)<sub>n residues</sub> + H<sub>2</sub>O → (DNA)<sub>n-1 residues</sub> + dNMP] and hence provides Pol I with the ability to '''proofread''' and '''edit''' its mistakes.<br />
<br />
:3. A 5' → 3' exonuclease, whose central role is to remove the RNA primers (although it also participates in DNA repair processes), which the polymerase function then replaces with DNA. <br />
<br />
These active sites occupy different regions of Pol I. In fact, mild treatment of Pol I by proteases such as trypsin and subtilisin, cleaves Pol I into two catalytically active fragments. The N-terminal fragment (residues 1-323) contains the 5' → 3' exonuclease function, whereas the larger, C-terminal fragment (residues 324-928), which is known as the '''Klenow fragment,''' contains both the polymerase and the 3' → 5' exonuclease functions.<br />
<br />
<applet load='1KLN_pymol.pdb' size='300' frame='true' align='right' caption='Klenow–DNA' scene='Sandbox_dvoet/DNA_polymerase/Klenow-dna/3' /><br />
<br />
Thomas Steitz determined the X-ray structure of Klenow fragment in complex with a 13-nucleotide (nt) primer strand and a 10-nt template strand (the primer strand is the strand that is synthesized by the polymerase as the complement of the template strand; the entire DNA is often referred to as primer−template DNA). Here Klenow fragment is shown in ribbon form colored in rainbow order from its N-terminus (''blue'') to its C-terminus (''red''). The DNA is drawn in stick form and colored according to atom type with template C cyan, primer C magenta, N blue, O red, and P orange and with an orange rod connecting successive P atoms in each strand. The 3' → 5' exonuclease active site at the N-terminal end of the protein is marked by a Zn<sup>2+</sup> ion (''gray sphere''). 'The arrangement of the polymerase's three domains is reminiscent of a right hand grasping a rod (the DNA) and hence, from N- to C-terminus, they are named “palm“, "fingers", and "thumb". The polymerase's active site is located in the palm domain near the cleft between the fingers and thumb domains. All DNA polymerases of known structure have a similar spatial arrangements of fingers, thumb, and palm domains, even though, in many cases, they have no recognizable sequence similarity with Pol I and the structure of their fingers, thumb, and palm domains bear no resemblance to those of Pol I. <br />
<br />
{{Clear}}<br />
<br />
<applet load='1KLN_pymol.pdb' size='300' frame='true' align='right' caption='Klenow–DNA closeup' scene='Sandbox_dvoet/DNA_polymerase/Klenow-dna-closeup/4' /><br />
<br />
The X-ray structure is that of an editing complex, that is, the 3' end of the primer strand, the end that is elongated by the polymerase, occupies the 3'→5' exonuclease active site. This is more clearly seen in a <scene name='Sandbox_dvoet/DNA_polymerase/Klenow-dna-closeup/3'>closeup of the DNA</scene> in which the the rods connecting successive P atoms have been removed for clarity. Note that the base pair closest to the polymerase active site, a G·C, has opened up to enable the 3' end of the primer strand to reach the exonuclease active site. Click here to <scene name='Sandbox_dvoet/DNA_polymerase/Dna-closeup/3'>hide the protein</scene>.</div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1017073Sandbox/Judy Voet/PFK2009-11-16T14:13:00Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-controlling enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P). <br />
The symmetry model of allosterism requires that an oligomeric (multisubunit) protein maintain its molecular symmetry in undergoing a transition from its high-activity R state to its low-activity T state. Hence, this transition must be concerted, with no intermediate states. The X-ray and enzymological evidence indicates that PFK, a tetramer of identical subunits, is an allosteric enzyme that follows the symmetry model. Hence, the binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. This kinemage shows the same two subunits of the tetramer as does. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
View1 shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are important participants in PFK's allosterically facilitated conformational change (see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
View2 is a closeup of the upper portion of View1 showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the adjacent active site. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the presence of the carboxylate group of Glu 161 have on the phosphate group of F6P were it to bind in the active site? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View2 for a closeup of the F6P-sidechain interactions.<br />
<br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4PFK; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K.<br />
<br />
==References==<br />
<ref group="xtra">PMID:6115424</ref><references group="xtra"/><br />
<ref group="xtra">PMID:2136935</ref><references group="xtra"/><br />
<ref group="xtra">PMID:10759544</ref><references group="xtra"/></div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1017061Sandbox/Judy Voet/PFK2009-11-16T10:39:53Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-controlling enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P). <br />
The symmetry model of allosterism requires that an oligomeric (multisubunit) protein maintain its molecular symmetry in undergoing a transition from its high-activity R state to its low-activity T state. Hence, this transition must be concerted, with no intermediate states. The X-ray and enzymological evidence indicates that PFK, a tetramer of identical subunits, is an allosteric enzyme that follows the symmetry model. Hence, the binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. This kinemage shows the same two subunits of the tetramer as does. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
View1 shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are important participants in PFK's allosterically facilitated conformational change (see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
View2 is a closeup of the upper portion of View1 showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the adjacent active site. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the presence of the carboxylate group of Glu 161 have on the phosphate group of F6P were it to bind in the active site? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View2 for a closeup of the F6P-sidechain interactions.<br />
<br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4PFK; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K.<br />
<br />
==References==<br />
<ref group="xtra">PMID:2136935 PMID: 10759544 </ref><references group="xtra"/></div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1017056Sandbox/Judy Voet/PFK2009-11-16T08:50:38Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/4pfk_biol/1' size='300' frame='true' align='right' caption='PFK: R-state Biological tetramer; generated from 4pfk by QPS' /><br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-controlling enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P). <br />
The symmetry model of allosterism requires that an oligomeric (multisubunit) protein maintain its molecular symmetry in undergoing a transition from its high-activity R state to its low-activity T state. Hence, this transition must be concerted, with no intermediate states. The X-ray and enzymological evidence indicates that PFK, a tetramer of identical subunits, is an allosteric enzyme that follows the symmetry model. Hence, the binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. This kinemage shows the same two subunits of the tetramer as does. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
View1 shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are important participants in PFK's allosterically facilitated conformational change (see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
View2 is a closeup of the upper portion of View1 showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the adjacent active site. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
== 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
This KINEMAGE shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. <br />
<kinemage align="right" width="450" height= "450" file="PFK2.kin" /><br />
KINEMAGE 2 comes up in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the presence of the carboxylate group of Glu 161 have on the phosphate group of F6P were it to bind in the active site? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View2 for a closeup of the F6P-sidechain interactions.<br />
<br />
{{clear}}<br />
== Atomic Coordinates==<br />
The atomic coordinates for R state PFK were obtained from 4PFK; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K.</div>Judy Voethttps://proteopedia.org/index.php?title=File:4pfk_biol.pdb&diff=1017054File:4pfk biol.pdb2009-11-16T08:21:38Z<p>Judy Voet: 4PFK biological molecule</p>
<hr />
<div>== Summary ==<br />
4PFK biological molecule<br />
== Licensing ==<br />
{{subst:Non-commercial from license selector}}</div>Judy Voethttps://proteopedia.org/index.php?title=File:PFK2.kin&diff=1017053File:PFK2.kin2009-11-16T08:18:24Z<p>Judy Voet: PFK kinemage 2</p>
<hr />
<div>== Summary ==<br />
PFK kinemage 2<br />
== Licensing ==<br />
{{subst:Non-commercial from license selector}}</div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1017052Sandbox/Judy Voet/PFK2009-11-16T08:11:04Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/Pfk-r_state/1' size='300' frame='true' align='right' caption='PFK: R-state; from 4pfk' /><br />
<br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-controlling enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P). <br />
The symmetry model of allosterism requires that an oligomeric (multisubunit) protein maintain its molecular symmetry in undergoing a transition from its high-activity R state to its low-activity T state. Hence, this transition must be concerted, with no intermediate states. The X-ray and enzymological evidence indicates that PFK, a tetramer of identical subunits, is an allosteric enzyme that follows the symmetry model. Hence, the binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme.<br />
<br />
This Kinemage exercise consists of two kinemage scenes that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==1. Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<br />
<br />
PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. This kinemage shows the same two subunits of the tetramer as does. <br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
View1 shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are important participants in PFK's allosterically facilitated conformational change (see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
View2 is a closeup of the upper portion of View1 showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the adjacent active site. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
==KINEMAGE 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
KINEMAGE 2 shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. KINEMAGE 2 comes up in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the presence of the carboxylate group of Glu 161 have on the phosphate group of F6P were it to bind in the active site? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View2 for a closeup of the F6P-sidechain interactions.<br />
<br />
<br />
The atomic coordinates for R state PFK were obtained from 4PFK; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K.<br />
<br />
CAPTION for Kinemage #1:<br />
R and T state phosphofructokinase PFK). R state subunits are pinktint and pink, and T state subunits are bluetint and skyblue. In the R state, F6P (hotpink) is bound to the active site, and ADP is bound both to the active site (green) and the allosteric site (yellow). In the T state, the allosteric inhibitor 2-phosphoglycolate (PGC; a PEP analog) is bound to the allosteric site (gold). The F6P-binding site in the T state, which does not bind PFK, is marked by the position of the F6P in the R state (gray). The side chains of Glu 161 and Arg 162 are red and cyan.<br />
<br />
CAPTION for Kinemage #2:<br />
The allosteric site of phosphofructokinase (PFK) with residues 53-60 not shown. Residues of R-state Subunits 1 and 2 are pinktint and pink, whereas T state residues of Subunits 1 and 2 are bluetint and skyblue. In the R state, active site-bound F6P is hotpink and allosteric site-bound ADP is yellow. In the T state, allosteric site-bound 2-phosphoglycolate (PGC) is gold. The T state active site, which does not contain F6P, is marked by the position of R state-bound F6P (gray; "F6P site"). The side chains of Glu 161 and Arg 162 are red and cyan.</div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1017051Sandbox/Judy Voet/PFK2009-11-16T07:40:51Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<applet load='4pfk' scene='Sandbox/Judy_Voet/PFK/Pfk-r_state/1' size='300' frame='true' align='right' caption='Collagen' /><br />
<br />
<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions (Table 14-1) and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-controlling enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P). <br />
The symmetry model of allosterism requires that an oligomeric (multisubunit) protein maintain its molecular symmetry in undergoing a transition from its high-activity R state to its low-activity T state. Hence, this transition must be concerted, with no intermediate states. The X-ray and enzymological evidence indicates that PFK, a tetramer of identical subunits, is an allosteric enzyme that follows the symmetry model. Hence, the binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme.<br />
<br />
This exercise consists of two kinemages that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
==Conformational Changes in a Dimeric Unit of PFK==<br />
<br />
<kinemage align="right" width="450" height= "450" file="PFK1.kin" /><br />
KINEMAGE 1: Conformational Changes in a Dimeric Unit of PFK.<br />
<br />
<br />
<br />
KINEMAGE 1: Conformational Changes in a Dimeric Unit of PFK.<br />
<br />
PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. This kinemage shows the same two subunits of the tetramer as does. View1 shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are important participants in PFK's allosterically facilitated conformational change (see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
View2 is a closeup of the upper portion of View1 showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the adjacent active site. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
==KINEMAGE 2: The Major Conformational Changes in a Subunit of PFK.==<br />
<br />
KINEMAGE 2 shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. KINEMAGE 2 comes up in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the presence of the carboxylate group of Glu 161 have on the phosphate group of F6P were it to bind in the active site? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View2 for a closeup of the F6P-sidechain interactions.<br />
<br />
<br />
The atomic coordinates for R state PFK were obtained from 4PFK; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K.<br />
<br />
CAPTION for Kinemage #1:<br />
R and T state phosphofructokinase PFK). R state subunits are pinktint and pink, and T state subunits are bluetint and skyblue. In the R state, F6P (hotpink) is bound to the active site, and ADP is bound both to the active site (green) and the allosteric site (yellow). In the T state, the allosteric inhibitor 2-phosphoglycolate (PGC; a PEP analog) is bound to the allosteric site (gold). The F6P-binding site in the T state, which does not bind PFK, is marked by the position of the F6P in the R state (gray). The side chains of Glu 161 and Arg 162 are red and cyan.<br />
<br />
CAPTION for Kinemage #2:<br />
The allosteric site of phosphofructokinase (PFK) with residues 53-60 not shown. Residues of R-state Subunits 1 and 2 are pinktint and pink, whereas T state residues of Subunits 1 and 2 are bluetint and skyblue. In the R state, active site-bound F6P is hotpink and allosteric site-bound ADP is yellow. In the T state, allosteric site-bound 2-phosphoglycolate (PGC) is gold. The T state active site, which does not contain F6P, is marked by the position of R state-bound F6P (gray; "F6P site"). The side chains of Glu 161 and Arg 162 are red and cyan.</div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1016949Sandbox/Judy Voet/PFK2009-11-15T16:03:59Z<p>Judy Voet: </p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<applet load='4pfk' scene='Collagen/Collagen_initial/1' size='300' frame='true' align='right' caption='Collagen' /><br />
<br />
<br />
<br />
<scene name='Collagen/1cag/2'>alanine</scene><br />
<br />
==Conformational Changes in a Dimeric Unit of PFK==<br />
<kinemage align="right" width="450" height="400" file="PFK1.kin" /><br />
<br />
KINEMAGE 1: Conformational Changes in a Dimeric Unit of PFK.<br />
KINEMAGE 2: The Major Conformational Changes in a Subunit of PFK.<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions (Table 14-1) and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-controlling enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P). <br />
The symmetry model of allosterism requires that an oligomeric (multisubunit) protein maintain its molecular symmetry in undergoing a transition from its high-activity R state to its low-activity T state. Hence, this transition must be concerted, with no intermediate states. The X-ray and enzymological evidence indicates that PFK, a tetramer of identical subunits, is an allosteric enzyme that follows the symmetry model. Hence, the binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme.<br />
<br />
This exercise consists of two kinemages that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
<br />
<br />
KINEMAGE 1: Conformational Changes in a Dimeric Unit of PFK.<br />
<br />
PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. This kinemage shows the same two subunits of the tetramer as does. View1 shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are important participants in PFK's allosterically facilitated conformational change (see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
View2 is a closeup of the upper portion of View1 showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the adjacent active site. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
KINEMAGE 2: The Major Conformational Changes in a Subunit of PFK.<br />
<br />
KINEMAGE 2 shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. KINEMAGE 2 comes up in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the presence of the carboxylate group of Glu 161 have on the phosphate group of F6P were it to bind in the active site? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View2 for a closeup of the F6P-sidechain interactions.<br />
<br />
<br />
The atomic coordinates for R state PFK were obtained from 4PFK; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K.<br />
<br />
CAPTION for Kinemage #1:<br />
R and T state phosphofructokinase PFK). R state subunits are pinktint and pink, and T state subunits are bluetint and skyblue. In the R state, F6P (hotpink) is bound to the active site, and ADP is bound both to the active site (green) and the allosteric site (yellow). In the T state, the allosteric inhibitor 2-phosphoglycolate (PGC; a PEP analog) is bound to the allosteric site (gold). The F6P-binding site in the T state, which does not bind PFK, is marked by the position of the F6P in the R state (gray). The side chains of Glu 161 and Arg 162 are red and cyan.<br />
<br />
CAPTION for Kinemage #2:<br />
The allosteric site of phosphofructokinase (PFK) with residues 53-60 not shown. Residues of R-state Subunits 1 and 2 are pinktint and pink, whereas T state residues of Subunits 1 and 2 are bluetint and skyblue. In the R state, active site-bound F6P is hotpink and allosteric site-bound ADP is yellow. In the T state, allosteric site-bound 2-phosphoglycolate (PGC) is gold. The T state active site, which does not contain F6P, is marked by the position of R state-bound F6P (gray; "F6P site"). The side chains of Glu 161 and Arg 162 are red and cyan.</div>Judy Voethttps://proteopedia.org/index.php?title=File:PFK1.kin&diff=1016948File:PFK1.kin2009-11-15T16:03:01Z<p>Judy Voet: </p>
<hr />
<div>== Licensing ==<br />
{{subst:Non-commercial from license selector}}</div>Judy Voethttps://proteopedia.org/index.php?title=Sandbox/Judy_Voet/PFK&diff=1016945Sandbox/Judy Voet/PFK2009-11-15T15:49:34Z<p>Judy Voet: New page: ==Phosphofructokinase (PFK)== <applet load='4pfk' scene='Collagen/Collagen_initial/1' size='300' frame='true' align='right' caption='Collagen' /> <scene name='Collagen/1cag/2'>alanine</...</p>
<hr />
<div>==Phosphofructokinase (PFK)==<br />
<applet load='4pfk' scene='Collagen/Collagen_initial/1' size='300' frame='true' align='right' caption='Collagen' /><br />
<br />
<br />
<br />
<scene name='Collagen/1cag/2'>alanine</scene><br />
<br />
==Conformational Changes in a Dimeric Unit of PFK==<br />
<kinemage align="right" width="450" height="400" file="E13_PFK_FOB3.kin" /><br />
<br />
KINEMAGE 1: Conformational Changes in a Dimeric Unit of PFK.<br />
KINEMAGE 2: The Major Conformational Changes in a Subunit of PFK.<br />
<br />
Phosphofructokinase (PFK) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). The PFK reaction is strongly exergonic (irreversible) under physiological conditions (Table 14-1) and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-controlling enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, and fructose-2,6-bisphosphate (F2,6P). <br />
The symmetry model of allosterism requires that an oligomeric (multisubunit) protein maintain its molecular symmetry in undergoing a transition from its high-activity R state to its low-activity T state. Hence, this transition must be concerted, with no intermediate states. The X-ray and enzymological evidence indicates that PFK, a tetramer of identical subunits, is an allosteric enzyme that follows the symmetry model. Hence, the binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme.<br />
<br />
This exercise consists of two kinemages that illustrate some of the allosterically-induced conformational changes that occur in PFK from Bacillus stearothermophilus.<br />
<br />
<br />
KINEMAGE 1: Conformational Changes in a Dimeric Unit of PFK.<br />
<br />
PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. This kinemage shows the same two subunits of the tetramer as does. View1 shows the two subunits in their R state conformation as represented by their Ca backbones with Subunit 1 in pinktint and Subunit 2 in pink. Two side chains in each subunit are shown, those of Glu 161 (red) and Arg 162 (cyan), which are important participants in PFK's allosterically facilitated conformational change (see below). An F6P (hotpink) and an ADP (green; "ADP-active") are bound in the active site of each subunit. An additional ADP (yellow; "ADP-allo") is bound in a separate so-called allosteric site of each subunit. The ADPs each have an associated Mg2+, which is represented here by a ball of the same color as the ADP to which it binds.<br />
<br />
Click the "ANIMATE" button to switch the dimer between its R and T states. In its T state, Subunit 1 is bluetint and Subunit 2 is skyblue. The side chains of Glu 161 and Arg 162 in both subunits are red and cyan as before (only the Ca and Cb atoms of the Arg 162 side chain in Subunit 1 are observed in the X-ray structure of the T state; those of Subunit 2 are all observed). The T state enzyme binds the inhibitor 2-phosphoglycolate (gold; "PGC"), a nonphysiological analog of the glycolytic intermediate phosphoenolpyruvate (PEP). Note that the binding site of PGC in the T state overlaps the allosteric binding site of ADP in the R state ("ADP-allo") and hence their binding is mutually exclusive. The T state active sites, which do not contain F6P, are marked by "ghost" F6Ps (gray;"F6P site"), which have the same positions as do the F6Ps in the R state enzyme.<br />
<br />
View2 is a closeup of the upper portion of View1 showing both the active site and the allosteric site in this region. Note that the active site is located at the interface between two subunits and that the allosteric site interacts directly with the adjacent active site. Compare the R state and T state conformations by displaying both at once or clicking on "ANIMATE". Can you identify the Mg2+ ion associated with each of the ADPs bound to the enzyme in the R state? Which ADP atoms coordinate these Mg2+ ions? <br />
<br />
The phosphate group of PGC binds to the allosteric site in the T state in very nearly the same position that the beta phosphate group of "ADP-allo" binds to the R state allosteric site; both phosphate groups bind to the side chains of the same three Arg residues (not shown).<br />
<br />
In the high-activity R state, the positively charged side chain of Arg 162 forms a hydrogen bonded salt bridge with the negatively charged 6-phosphate group of F6P (white dashed lines), an interaction which presumably stabilizes the R state relative to the T state and is therefore in part responsible for F6P's homotropic effect.<br />
<br />
KINEMAGE 2: The Major Conformational Changes in a Subunit of PFK.<br />
<br />
KINEMAGE 2 shows those segments near the allosteric site (residues 53-60 are not shown here). As in KINEMAGE 1, the polypeptide is represented by its Ca chain with R state Subunits 1 and 2 in redtint and pink, and T state Subunits 1 and 2 in bluetint and skyblue. KINEMAGE 2 comes up in the R state showing the phosphate group of F6P (hotpink) bound in the enzyme's active site in a hydrogen bonded salt bridge (dashed white lines) with the side chain of Arg 162 (cyan). An ADP (yellow; "ADP-allo") occupies the adjacent allosteric site. Click once on "ANIMATE" to switch to the T state. This replaces the ADP in the R state allosteric site with the inhibitor and PEP analog PGC (gold). F6P no longer occupies the active site but its position in the R state is indicated by the "ghost" F6P (gray; "F6P site"). <br />
<br />
What happens to the central polypeptide helical segment (residues 149-164) in the R to T transition? What does this do to the relative positions of the negatively charged Glu 161 and the positively charged Arg 162? Click on "F6P site". What influence would the presence of the carboxylate group of Glu 161 have on the phosphate group of F6P were it to bind in the active site? Does this explain, at least in part, why T state PFK has low affinity for F6P? Go to View2 for a closeup of the F6P-sidechain interactions.<br />
<br />
<br />
The atomic coordinates for R state PFK were obtained from 4PFK; those for T state PFK were obtained from Philip Evans, MRC Laboratory of Molecular Biology, Cambridge, U. K.<br />
<br />
CAPTION for Kinemage #1:<br />
R and T state phosphofructokinase PFK). R state subunits are pinktint and pink, and T state subunits are bluetint and skyblue. In the R state, F6P (hotpink) is bound to the active site, and ADP is bound both to the active site (green) and the allosteric site (yellow). In the T state, the allosteric inhibitor 2-phosphoglycolate (PGC; a PEP analog) is bound to the allosteric site (gold). The F6P-binding site in the T state, which does not bind PFK, is marked by the position of the F6P in the R state (gray). The side chains of Glu 161 and Arg 162 are red and cyan.<br />
<br />
CAPTION for Kinemage #2:<br />
The allosteric site of phosphofructokinase (PFK) with residues 53-60 not shown. Residues of R-state Subunits 1 and 2 are pinktint and pink, whereas T state residues of Subunits 1 and 2 are bluetint and skyblue. In the R state, active site-bound F6P is hotpink and allosteric site-bound ADP is yellow. In the T state, allosteric site-bound 2-phosphoglycolate (PGC) is gold. The T state active site, which does not contain F6P, is marked by the position of R state-bound F6P (gray; "F6P site"). The side chains of Glu 161 and Arg 162 are red and cyan.</div>Judy Voethttps://proteopedia.org/index.php?title=File:E13_PFK_FOB3.kin&diff=1016944File:E13 PFK FOB3.kin2009-11-15T15:46:04Z<p>Judy Voet: </p>
<hr />
<div>== Licensing ==<br />
{{subst:Non-commercial from license selector}}</div>Judy Voethttps://proteopedia.org/index.php?title=Proteopedia:Table_of_Contents&diff=1016943Proteopedia:Table of Contents2009-11-15T15:33:33Z<p>Judy Voet: </p>
<hr />
<div>{{#tree:id=siteTree|openlevels=1|root='''Chemistry of Life'''|close=top|open=top|<br />
<br />
* INTRODUCTION<br />
** Introduction to the Chemistry of Life<br />
<br />
** WATER<br />
<br />
*** [[Water_in_macromolecular_models|Water in Macromolecular Models]]<br />
** KEY CHEMICAL CONCEPTS FOR STRUCTURAL BIOLOGY<br />
<br />
*** [[Cation-pi interactions]]<br />
*** [[Hydrogen bond]]<br />
*** [[Hydrogen_in_macromolecular_models|Hydrogen in Macromolecular Models]]<br />
*** [[Isoelectric_point]]<br />
<br />
* BIOMOLECULES<br />
**[[About_Macromolecular_Structure]]<br />
<br />
** NUCLEOTIDES, NUCLEIC ACIDS AND GENETIC INFORMATION<br />
*** DNA<br />
**** [[DNA]]<br />
**** B-DNA [[1bna]]<br />
**** [[Z-DNA]]<br />
<br />
** AMINO ACIDS<br />
*** [[Amino_Acids]]<br />
*** [[Selenocysteine]]<br />
** PROTEINS: PRIMARY STRUCTURE<br />
*** [[Conservation,_Evolutionary]]<br />
*** [[Isoelectric_point]]<br />
<br />
<br />
** PROTEINS: THREE-DIMENSIONAL STRUCTURE<br />
*** [[S347/The four tiers of protein structure]]<br />
*** [[Secondary_structure]]<br />
*** Protein structural motifs<br />
**** [[User:James_D_Watson/Structural_Templates]]<br />
*** FIBROUS PROTEINS<br />
**** [[Coiled_coil]]<br />
**** [[Collagen]]<br />
*** Protein Misfolding<br />
**** [[Prion_protein]]<br />
**** [[A_Physical_Model_of_the_Structure_of_GNNQQNY_from_Yeast_Prion_Sup35]]<br />
<br />
** COLORED PROTEINS & THOSE THAT CHANGE COLOR<br />
*** [[Green Fluorescent Protein]]; [[GFP_(Hebrew)]]<br />
*** [[Dronpa]]<br />
*** [[Factor IX]]<br />
<br />
** DNA BINDING PROTEINS<br />
*** [[Helix-turn-helix motif]]<br />
*** [[DNA-binding protein VirE2 from Agrobacterium tumefaciens complexed with chaperone VirE1]]<br />
<br />
** PROTEIN FUNCTION: Myoglobin and Hemoglobin, Muscle Contraction, and Antibodies<br />
*** Antibodies<br />
**** [[IgA]]<br />
**** [[Epitopes]]<br />
**** [[Major_Histocompatibility_Complex_Class_I]]<br />
*** [[Calmodulin_in_motion]]<br />
*** [[Myoglobin]]<br />
*** [[Hemoglobin]]; [[Hemoglobin_(Hebrew)]]<br />
*** [[Kinesin-5]]<br />
<br />
** LIPIDS AND BIOLOGICAL MEMBRANES<br />
<br />
** MEMBRANE TRANSPORT PROTEINS<br />
*** [[A Physical Model of the β2-Adrenergic Receptor]]<br />
*** [[Lactose_Permease]]<br />
*** [[Proton_Channels]]<br />
*** [[Ion_channels]]<br />
*** [[Mechanosensitive_channels:_opening_and_closing]]<br />
***[[Enzyme_I_of_the_Phosphoenolpyruvate:Sugar_Phosphotransferase_System]]<br />
<br />
** PRIONS AND INTRINSICALLY DISORDERED PROTEINS<br />
*** [[Prion_protein]]<br />
*** [[Doppel]]<br />
*** [[Intrinsically Disordered Protein]]<br />
<br />
** TOXINS<br />
*** [[Insecticidal delta-endotoxin Cyt2Ba from Bacillus thuringiensis]]<br />
<br />
** MITOSIS, MEOSIS, AND CARGO TRANSPORT PROTEINS<br />
*** [[Kinesin-5]]<br />
<br />
** VIRUSES<br />
*** [[User:Wayne_Decatur/Suppression_of_RNA_Silencing_by_Viruses]]<br />
*** Filamentous bacteriphage<br />
**** [[G3p]] - minor coat protein found on the surface of filamentous bacteriophage<br />
*** HIV<br />
**** [[HIV-1 protease]]<br />
**** [[HIV-1 Gag]]<br />
**** [[HIV-1 Gag Recruitment of Tsg101 and the Viral Budding Process]]<br />
**** [[User:Eric_Martz/Molecular_Playground/HIVDrug]]<br />
*** Herpes Simplex Virus<br />
**** [[Herpes_Simplex_Virus_Thymidine_Kinase]]<br />
*** Influenza<br />
**** [[Influenza_hemagglutinin]]<br />
<br />
* ENZYMES<br />
** Enzymatic Catalysis<br />
*** [[Aconitase]]<br />
*** [[Enzyme I of the Phosphoenolpyruvate:Sugar Phosphotransferase System]]<br />
*** Serine Hydrolases<br />
**** [[Serine_Protease]]<br />
**** [[Trypsin]]<br />
**** [[Alpha-1-antitrypsin]]<br />
**** [[Acetylcholinesterase]] (AChE)<br />
***** [[Acetylcholine]]<br />
***** [[Flexibility_of_aromatic_residues_in_acetylcholinesterase]]<br />
***** [[AChE inhibitors and substrates]]<br />
****** [[1eve]] AChE-Aricept complex; [[1eve (Chinese)]]; [[1eve (Russian)]]; [[1eve (Spanish)]]; [[1eve (Turkish)]]<br />
****** [[AChE_bivalent_inhibitors]]<br />
*** [[Triose_Phosphate_Isomerase]]<br />
*** [[Pyruvate_phosphate_dikinase]]<br />
*** Cysteine Proteases<br />
**** [[Tobacco_Etch_Virus_(TEV)_Protease]]<br />
**** [[Streptomyces_griseus_Aminopeptidase_(SGAP)]]; [[Aminopeptidase]]<br />
*** Acid Proteases<br />
**** [[HIV-1 protease]]<br />
**** [[Pepsin]]<br />
*** Metaloproteases<br />
**** [[Metalloproteases]]<br />
**** [[Matrix_metalloproteinases]]<br />
*** Oxidoreductases<br />
**** [[NADH quinone oxidoreductase ]]<br />
<br />
** Enzyme Kinetics, Inhibition, and Control<br />
<br />
** BIOCHEMICAL SIGNALING<br />
*** [[Nitric_oxide_synthase]]<br />
*** [[Recoverin,_a_calcium-activated_myristoyl_switch]]<br />
*** [[Bcl-2]]<br />
*** [[C-Myc]]<br />
<br />
* METABOLISM<br />
** Introduction to Metabolism<br />
<br />
** Glucose Catabolism<br />
*** [[Triose_Phosphate_Isomerase]]<br />
** Glycogen Metabolism and Gluconeogenesis<br />
*** [[Phosphoglucose_isomerase]]<br />
*** [[Calmodulin_in_motion]]<br />
*** [[Biotin_Protein_Ligase]]<br />
<br />
** Citric Acid Cycle<br />
*** [[Aconitase]]<br />
<br />
** Electron Transport and Oxidative Phosphorylation<br />
*** [[NADH quinone oxidoreductase ]]<br />
<br />
** Photosynthesis<br />
*** [[Photosystem_II]]<br />
*** [[Ribulose-1,5-bisphosphate_carboxylase/oxygenase]]<br />
*** [[PrrA_in_Rhodobacter_sphaeroides]]<br />
*** [[Pyruvate_phosphate_dikinase]]<br />
<br />
** Lipid Metabolism<br />
*** [[Acid-beta-glucosidase]]<br />
** Amino Acid Metabolism<br />
*** [[Aromatic_amino_acid_hydroxylases]]<br />
*** [[Phenylalanine_hydroxylase]]<br />
*** [[Tyrosine_hydroxylase]]<br />
*** [[ATP_Phosphoribosyl_Transferase]]<br />
*** [[Isochorismate_pyruvate_lyase]]<br />
** Mammalian Fuel Metabolism: Integration and Regulation<br />
** Nucleotide Metabolism<br />
*** [[Dihydrofolate_reductase]]<br />
<br />
* GENE EXPRESSION AND REPLICATION <br />
<br />
<br />
** Nucleic Acid Structure<br />
*** [[DNA]]<br />
**** B-DNA [[1bna]]<br />
**** [[Z-DNA]]<br />
*** [[Nucleosomes]]<br />
** DNA Replication, Repair, and Recombination<br />
*** [[DNA Replication,Transcription and Translation]]<br />
*** [[PcrA_helicase]]<br />
*** [[Fpg_Nei_Protein_Superfamily]] - DNA Repair and Base Excision DNA Repair<br />
*** [[Human RecQ-Like protein 1]] - RecQ family of DNA helicases are conserved in from bacteria to man <br />
*** [[Structure_of_E._coli_DnaC_helicase_loader]]<br />
***[[1x9n#Crystal_Structure_of_Human_DNA_Ligase_I_bound_to_5.27-adenylated.2C_nicked_DNA]]<br />
*** [[Rop_protein]]<br />
<br />
** Transcription and RNA Processing<br />
*** [[C-Myc]]<br />
*** [[Lac_repressor]]<br />
*** [[TATA-Binding_Protein]]<br />
*** [[RSP1275]]<br />
*** [[Transcription_Termination_Factor_Rho]]<br />
<br />
** Protein Synthesis<br />
*** [[Ribosome]]<br />
*** [[SelB_Recognition]]<br />
** Regulation of Gene Expression<br />
*** [[Lac_repressor]]<br />
*** [[Irr]]<br />
*** [[P53]]<br />
*** [[Tangible_Models_of_Cdc42_Interacting_With_Intersectin]]<br />
<br />
* EVOLUTION<br />
** [[Conservation, Evolutionary]]<br />
<br />
** [[Extremophiles]]<br />
<br />
* IMMUNE SYSTEM<br />
** Antibodies<br />
*** [[IgA]]<br />
*** [[Epitopes]]<br />
** [[Major_Histocompatibility_Complex_Class_I]]<br />
<br />
<br />
<br />
* METHODS OF STRUCTURE DETERMINATION <br />
** X-ray<br />
*** [[X-ray_crystallography]]<br />
**** [[Asymmetric_Unit]]<br />
**** [[Biological_Unit]]<br />
**** [[Electron_density_maps]]<br />
*** SAXS<br />
** NMR<br />
*** [[NMR_Ensembles_of_Models]]<br />
** Electron Microscopy<br />
<br />
* METHODS OF STRUCTURE & SEQUENCE ANALYSIS <br />
** STRUCTURAL ANALYSIS & VISUALIZATION<br />
*** [[Chime]]<br />
*** Jmol<br />
**** [[S347/Visualising_protein_structure|Visualising Protein Structure]] - Introduction to Jmol<br />
**** [[FirstGlance_in_Jmol]]<br />
*** [[User:Wayne Decatur/Teaching Proteopedia|Teaching Proteopedia]]<br />
*** [[User:Wayne Decatur/Generate Unfolded Structures|Generate Unfolded Structures]]<br />
*** Homology Model<br />
**** [[User:Wayne Decatur/Homology Modeling|Homology Modeling]]<br />
**** [[User:Emi Nakayama/TRIM5a Homology Models|TRIM5a Homology Models]] - Differences in models as a function of their templates<br />
*** [[DRuMS]] - set of standard color schemes for macromolecular visualization<br />
<br />
** SEQUENCE ANALYSIS<br />
*** [[User:Wayne Decatur/Sequence analysis tools|Sequence Analysis Tools]]<br />
<br />
}}</div>Judy Voet