{{STRUCTURE_1pfk | PDB=1pfk | SCENE= }}
Phosphofructokinase (PFK) (PDB id [[4pfk]]) is an approximately 300 residue enzyme that catalyzes the phosphorylation of Fructose-6-phosphate (F6P) to Fructose-1,6-bisphosphate (F1,6P) in the third reaction of glycolysis. This enzyme, with four subunits, catalyzes the most highly regulated reaction of glycolysis. Phosphofructokinase is not only the enzymes name, but also, the fold, superfamily, and family classification name.

==Role in Glycolysis==
Glycolysis is the process of breaking down glucose to make pyruvic acid, which is used in anaerobic respiration or as one of the starting reactants in the citric acid cycle. The process releases some energy but more importantly paves the way for vast amounts of energy to be made through the citric acid cycle. After glucose has been phosphorylated and isomerized to Fructose-6-phospate, PFK begins its work. It phosphorylates the hydroxy group at the number one carbon, which was impossible in glucose. This second phosphorylation by PFK is important because it makes a doubly-high-energy compound. High energy compounds, like Fructose-6-phosphate and Fructose-1,6-bisphosphate, help to drive the endergonic processes of glycolysis through their own exergonic breakdown.<ref>Voet, Donald, Judith G. Voet, and Charlotte W. Pratt. Fundamentals of Biochemistry: Life at the Molecular Level. Hoboken, NJ: Wiley, 2008. Print.</ref>. It is important then that PFK makes a bisphosphate compound, because eventually that molecule will be cut in half. Thus, after the action of PFK, the six-carbon compound can be broken into two high-energy three-carbon compounds, which are both ready to move onto the next steps of glycolysis.

==Structure==
PFK is a <scene name='Zach_Westrick_Sandbox/Tetramer/1'>Homotetramer</scene> that acts as a dimer of dimers, similar to hemoglobin.<ref>PMID:6115424</ref>. One half of each dimer is involved in the binding of ATP, while the other is involved with substrate binding and also contains an allosteric site. <ref>PMID:2975709</ref>. One subunit with ATP and F6P bound can be seen below.
PFK's <scene name='Zach_Westrick_Sandbox/Secondary_structure/3'>Secondary Structure</scene> puts it in an alpha and beta class. Each unit is comprised of two domains that sandwich parallel beta sheets in between alpha helices. The outer most beta sheets of the larger domain, however, are anti-parallel. This is best seen in the <scene name='Zach_Westrick_Sandbox/One_subunit/1'>4pfk image</scene>.

[[Image:Subunit.JPG]]

==Mechanism==
Phosphofructokinase binds both Mg2+-ATP and fructose-6-phosphate (F6P) to make fructose-1,6-bisphosphate and Mg2+-ADP. Although the image with both of these products has not been determined, <scene name='Zach_Westrick_Sandbox/Products_2/2'>F6P and Mg2+-ADP</scene> bound to the enzyme has been. There are three ligand binding sites per subunit. Two make up the active site, which binds F6P and ATP, while the third is an allosteric binding site.<ref>PMID:6115424</ref> Some proposed residues involved at the active site include <scene name='Zach_Westrick_Sandbox/Active_site_2/1'>ASP 127 and ARG 171</scene>.<ref>http://www.nature.com/nature/journal/v327/n6121/abs/327437a0.html</ref> PFK exist in two conformational states, both <scene name='Zach_Westrick_Sandbox/R_state/1'>R</scene> and <scene name='Zach_Westrick_Sandbox/T_state/1'>T states</scene> which are in equilibrium. ATP binds both active and allosteric sites in both conformations.

[[Image:PFK mech.JPG]]

==Regulation==
Glycolysis is an essential metabolic process for survival. Therefore, in its activation and suppression it must be highly regulated. Three points in the process of glycolysis occur with a large negative free energy and are therefore, irreversible. These three points are hexokinase, phosphofructokinase, and pyruvate kinase. These three reactions are candidates to be the major points of regulation because of their high negative free energies. Of the three PFK is considered the major regulatory point for glycolysis (#3 in the picture below) in muscle with a ΔG= -25.9 kJ/mol. <ref>Voet, Donald, Judith G. Voet, and Charlotte W. Pratt. Fundamentals of Biochemistry: Life at the Molecular Level. Hoboken, NJ: Wiley, 2008. Print.</ref>. This makes sense seeing as pyruvate kinase catalyzes the final reaction (#10) and hexokinase (#1) is not involved in glycolysis at all when the process is begun from glycogen.<ref>Voet, Donald, Judith G. Voet, and Charlotte W. Pratt. Fundamentals of Biochemistry: Life at the Molecular Level. Hoboken, NJ: Wiley, 2008. Print.</ref>

[[Image:Paint_image.JPG]]

PFK is regulated by ATP, AMP, ADP. While ATP binds at the active site equally well in both R and T states, it preferentially binds the allosteric site of the T state <ref>Voet, Donald, Judith G. Voet, and Charlotte W. Pratt. Fundamentals of Biochemistry: Life at the Molecular Level. Hoboken, NJ: Wiley, 2008. Print.</ref> This preferential binding causes a shift from equilibrium of the two states, to a greater amount of T state <ref>PubMed:2136935</ref>, which decreases the affinity for F6P. Allosteric activator <scene name='Zach_Westrick_Sandbox/Allosteric_activator/2'>ADP</scene> also binds to allosteric site to increase the ratio of R state phosphofructokinase. As can be seen from the graph below, the plots for the activity of PFK are sigmoidal. This further demonstrates the cooperative nature of the enzyme. The initial binding of substrate to the enzyme is difficult, but once it has and forces the change in state from T -> R the other substrates bind much more easily. The graph also shows that adding ATP moves the plot right (ie decreases affinity for F6P), while adding AMP moves it to the left.

[[Image:Inhib.jpg]]

The system of regulation matches well with the function of PFK. When PFK is active, ATP is being produced down stream from it as glucose is eventually broken down completely. Thus, when ATP levels are low and more needs to be made, the activity of PFK will be increased, because ADP will be in high concentration. The opposite holds true as well, when ATP is in high concentration and inhibits protein activity. And yet, this explanation cannot completely account for the regulation of PFK, because the levels of ATP do not very greatly enough between active and resting muscles. Another means of allosteric regulation must be found.<ref>Voet, Donald, Judith G. Voet, and Charlotte W. Pratt. Fundamentals of Biochemistry: Life at the Molecular Level. Hoboken, NJ: Wiley, 2008. Print.</ref>

The PFK's Km for ATP is .020mM and .032mM.<ref>PMID: 6233271</ref>

==Additional Resources==
For additional information, see: [[Carbohydrate Metabolism]]
 

==References==
<references/>

File:Inhib.jpg

2011-04-12T19:15:27Z

Robert Cassady:

2011-04-12T18:28:57Z

Robert Cassady: /* Structure */

Cassady sandbox1

2011-04-12T18:27:01Z

Robert Cassady: /* Role in Glycolysis */

{{STRUCTURE_1pfk | PDB=1pfk | SCENE= }}
Phosphofructokinase (PFK) (PDB id [[4pfk]]) is an approximately 300 residue enzyme that catalyzes the phosphorylation of Fructose-6-phosphate (F6P) to Fructose-1,6-bisphosphate (F1,6P) in the third reaction of glycolysis. This enzyme, with four subunits, catalyzes the most highly regulated reaction of glycolysis.

==Role in Glycolysis==
Glycolysis is the process of breaking down glucose to make pyruvic acid, which is used in anaerobic respiration or as one of the starting reactants in the citric acid cycle. The process releases some energy but more importantly paves the way for vast amounts of energy to be made through the citric acid cycle. After glucose has been phosphorylated and isomerized to Fructose-6-phospate, PFK begins its work. It phosphorylates the hydroxy group at the number one carbon, which was impossible in glucose. This second phosphorylation by PFK is important because it makes a doubly-high-energy compound. High energy compounds, like Fructose-6-phosphate and Fructose-1,6-bisphosphate, help to drive the endergonic processes of glycolysis through their own exergonic breakdown.<ref>Voet, Donald, Judith G. Voet, and Charlotte W. Pratt. Fundamentals of Biochemistry: Life at the Molecular Level. Hoboken, NJ: Wiley, 2008. Print.</ref>. It is important then that PFK makes a bisphosphate compound, because eventually that molecule will be cut in half. Thus, after the action of PFK, the six-carbon compound can be broken into two high-energy three-carbon compounds, which are both ready to move onto the next steps of glycolysis.

==Structure==
It is a <scene name='Zach_Westrick_Sandbox/Tetramer/1'>Homotetramer</scene> that acts as a dimer of dimers, similar to hemoglobin.<ref>PMID:6115424</ref>. One half of each dimer is involved in the binding of ATP, while the other is involved with substrate binding and also contains an allosteric site. <ref>PMID:2975709</ref>. One subunit with ATP and F6P bound can be seen below. The <scene name='Zach_Westrick_Sandbox/Secondary_structure/3'>Secondary Structure</scene> puts it in an alpha and beta class. Each unit is comprised of two domains that sandwich parallel beta sheets in between alpha helices. The outer most beta sheets of the larger domain, however, are anti-parallel. This is best seen in the <scene name='Zach_Westrick_Sandbox/One_subunit/1'>4pfk image</scene>. Phosphofructokinase is not only the enzymes name, but also, the fold, superfamily, and family classification name.

[[Image:Subunit.JPG]]

==Mechanism and Regulation of Phosphofructokinase==
Phosphofructokinase binds both Mg2+-ATP and fructose-6-phosphate (F6P) to make fructose-1,6-bisphosphate and Mg2+-ADP. Although the image with both of these products has not been determined, <scene name='Zach_Westrick_Sandbox/Products_2/2'>F6P and Mg2+-ADP</scene> bound to the enzyme has been. There are three ligand binding sites per subunit. Two make up the active site, which binds F6P and ATP, while the third is an allosteric binding site.<ref>PMID:6115424</ref> Some proposed residues involved at the active site include <scene name='Zach_Westrick_Sandbox/Active_site_2/1'>ASP 127 and ARG 171</scene>.<ref>http://www.nature.com/nature/journal/v327/n6121/abs/327437a0.html</ref> PFK exist in two conformational states, both <scene name='Zach_Westrick_Sandbox/R_state/1'>R</scene> and <scene name='Zach_Westrick_Sandbox/T_state/1'>T states</scene> which are in equilibrium. ATP binds both active and allosteric sites in both conformations. While ATP binds the active site equally well, it preferentially binds the allosteric site of the T state <ref>Voet, Donald, Judith G. Voet, and Charlotte W. Pratt. Fundamentals of Biochemistry: Life at the Molecular Level. Hoboken, NJ: Wiley, 2008. Print.</ref> This preferential binding causes a shift from equilibrium of the two states, to a greater amount of T state <ref>PubMed:2136935</ref>, which decreases the affinity for F6P. Allosteric activator <scene name='Zach_Westrick_Sandbox/Allosteric_activator/2'>ADP</scene> also binds to allosteric site to increase the ratio of R state phosphofructokinase. Along with ADP,AMP and F2,6P inhibit the regulatory role of ATP. Three points in the process of glycolysis occur with a large negative free energy and are therefore, irreversible. These three points are hexokinase, phosphofructokinase, and pyruvate kinase; of these three PFK is considered the major regulatory point for glycolysis in muscle with a ΔG= -25.9 kJ/mol. <ref>Voet, Donald, Judith G. Voet, and Charlotte W. Pratt. Fundamentals of Biochemistry: Life at the Molecular Level. Hoboken, NJ: Wiley, 2008. Print.</ref>

The PFK's Km for ATP is .020mM and .032mM.<ref>PMID: 6233271</ref>

[[Image:PFK mech.JPG]] [[Image:Paint_image.JPG]][[Image:Inhib.JPG]]

==Additional Resources==
For additional information, see: [[Carbohydrate Metabolism]]
 

==References==
<references/>

Cassady sandbox1

2011-04-12T18:23:00Z

2011-04-02T01:25:53Z

Robert Cassady: New page: ==Phosphofructokinase== Phosphofructokinase (PFK) (PDB id 4pfk) is an approximately 300 residue enzyme that catalyzes the phosphorylation of Fructose-6-phosphate (F6P) to Fructose-1,6-bisp...

==Phosphofructokinase==
Phosphofructokinase (PFK) (PDB id 4pfk) is an approximately 300 residue enzyme that catalyzes the phosphorylation of Fructose-6-phosphate (F6P) to Fructose-1,6-bisphosphate (F1,6P). It is a Homotetramer that acts as a dimer of dimers, similar to hemoglobin.[1] One half of each dimer is involved in the binding of ATP, while the other is involved with substrate binding and also contains an allosteric site. [2] The Secondary Structure puts it in an alpha and beta class. Each unit is comprised of two domains that sandwich parallel beta sheets inbetween alpha helices. The outer most beta sheets of the larger domain, however, are anti-parallel. This is best seen in the 4pfk image Phosphofructokinase is not only the enzymes name, but also, the fold, superfamily, and family classification name. While PFK in glycolysis is an ATP-dependent phosphofructokinase, Pyrophosphate-dependent phosphofructokinases exist as well.
Contents
[hide]

1 Role in Glycolysis
2 Mechanism and Regulation of Phosphofructokinase
3 Additional Resources
4 References

[edit] Role in Glycolysis

Glycolysis is the process for preparing, and breaking down, glucose to make pyruvic acid, which is used in anaerobic respiration or as one of the starting reactants in the citric acid cycle. Three points in the process of glycolysis occur with a large negative free energy and are therefore, irreversible. These three points are hexokinase, phosphofructokinase, and pyruvate kinase; of these three PFK is considered the major regulatory point for glycolysis in muscle with a ΔG= -25.9 kJ/mol. [3]
[edit] Mechanism and Regulation of Phosphofructokinase

Phosphofructokinase binds both Mg2+-ATP and fructose-6-phosphate (F6P) to make fructose-1,6-bisphosphate and Mg2+-ADP. Although the image with both of these products has not been determined, F6P and Mg2+-ADP bound to the enzyme has been. There are three ligand binding sites per subunit. Two make up the active site, which binds F6P and ATP, while the third is an allosteric binding site.[4] Some proposed residues involved at the active site include ASP 127 and ARG 171.[5] PFK exist in two conformational states, both R and T states which are in equilibrium. ATP binds both active and allosteric sites in both conformations. While ATP binds the active site equally well, it preferentially binds the allosteric site of the T state [6] This preferential binding causes a shift from equilibrium of the two states, to a greater amount of T state [7], which decreases the affinity for F6P. Allosteric activator ADP also binds to allosteric site to increase the ratio of R state phosphofructokinase. Along with ADP,AMP and F2,6P inhibit the regulatory role of ATP. The PFK's Km for ATP is .020mM and .032mM.[8]

Image:PFK mech.JPG
[edit] Additional Resources

For additional information, see: Carbohydrate Metabolism
[edit] References

↑ Evans PR, Farrants GW, Hudson PJ. Phosphofructokinase: structure and control. Philos Trans R Soc Lond B Biol Sci. 1981 Jun 26;293(1063):53-62. PMID:6115424
↑ Shirakihara Y, Evans PR. Crystal structure of the complex of phosphofructokinase from Escherichia coli with its reaction products. J Mol Biol. 1988 Dec 20;204(4):973-94. PMID:2975709
↑ Voet, Donald, Judith G. Voet, and Charlotte W. Pratt. Fundamentals of Biochemistry: Life at the Molecular Level. Hoboken, NJ: Wiley, 2008. Print.
↑ Evans PR, Farrants GW, Hudson PJ. Phosphofructokinase: structure and control. Philos Trans R Soc Lond B Biol Sci. 1981 Jun 26;293(1063):53-62. PMID:6115424
↑ http://www.nature.com/nature/journal/v327/n6121/abs/327437a0.html
↑ Voet, Donald, Judith G. Voet, and Charlotte W. Pratt. Fundamentals of Biochemistry: Life at the Molecular Level. Hoboken, NJ: Wiley, 2008. Print.
↑ PubMed:2136935
↑ Campos G, Guixe V, Babul J. Kinetic mechanism of phosphofructokinase-2 from Escherichia coli. A mutant enzyme with a different mechanism. J Biol Chem. 1984 May 25;259(10):6147-52. PMID:6233271

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