Proteopedia - User contributions [en]

Test 3625

2012-03-11T09:40:45Z

Harry Greenblatt:

<Structure load='4ab1' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />

First switch to <scene name='Test_3625/Scene_1/1'>rockets</scene>.
Now, colour <scene name='Test_3625/Scene_2/1'>Ser221</scene> red and ball and stick, zoomed.

Test 3625

2012-03-11T09:34:01Z

Harry Greenblatt:

<Structure load='4ab1' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />

First switch to <scene name='Test_3625/Scene_1/1'>rockets</scene>.

Test 3625

2012-03-11T09:31:17Z

Harry Greenblatt:

Test 3625

2012-03-11T09:30:27Z

Harry Greenblatt:

Test 3625

2012-03-11T09:23:41Z

Harry Greenblatt:

<Structure load='<Structure load='4ab1' size='500' frame='true' align='right' caption='test' scene='
<scene name='Test_3625/Scene_1/1'>TextToBeDisplayed</scene>' />' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />

Test 3625

2012-03-11T08:42:06Z

Harry Greenblatt: test of new jmol

<Structure load='<Structure load='4ab1' size='500' frame='true' align='right' caption='test' scene='Insert optional scene name here' />' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />

Beta-Hexosaminidase

2011-02-17T10:45:24Z

Harry Greenblatt:

<applet load="" size="600" color="" frame="true" spin="on" Scene ="Beta-Hexosaminidase/Opening/3" align="right" caption="Crystal Structure of Human β-Hexosaminidase, ([[2gjx]])"/>
===Structure of Human β-Hexosaminidase A and its association with Tay-Sachs disease===
<hr/>
β-Hexosaminidase A is a lysosomal enzyme essential for the degradation of GM2 gangliosides. Deficiency of lysosomal β-Hexosaminidase A due to inherited defects in the α-subunit gene results in Tay-Sachs (TS) disease. The 3D structure of β-Hexosaminidase A was determined by the group of Michael N.G. James at the University of Alberta, Edmonton, Canada.<ref>PMID:16698036</ref> The structure reveals an <scene name='Beta-Hexosaminidase/Subunits/4'>αβ-heterodimer</scene>, with each subunit having a functional active site. Only the <scene name='Beta-Hexosaminidase/Alpha/2'>α-subunit</scene> active site can hydrolyze GM2 gangliosides due to <scene name='Beta-Hexosaminidase/Gsep/6'>a flexible loop</scene> α280GSEP283 structure that is removed post-translationaly from β, and to the presence of <scene name='Beta-Hexosaminidase/4234/5'>α-Asn 423 and α-Arg 424</scene>. The <scene name='Beta-Hexosaminidase/Gsep_out/3'>loop structure</scene> is involved in binding the GM2 activator protein, while <scene name='Beta-Hexosaminidase/424_b/1'>α-Arg424</scene> is critical for binding the carboxylate group of the N-acetyl-neuraminic acid residue of GM2. <scene name='Beta-Hexosaminidase/2_active/1'>Two active sites</scene> are present in the HexA dimer; one comprising residues from <scene name='Beta-Hexosaminidase/Active_alpha/1'>the α-subunit</scene> (R178 D207 H262 E323 D322 W373 W392 W460 Y421 R424 N423 E462) and a second one from residues of the <scene name='Beta-Hexosaminidase/Active_beta/1'>β-subunit</scene> (R211 D240 H294 E355 D354 W405 W424 Y450 L453 D452 E491 W489). These active sites are located at the opening of TIM barrels at the interface between the α and β-subunits. The HexA <scene name='Beta-Hexosaminidase/Glyco/1'>undergoes glycosylation</scene> on the α and β-subunits; α-Asn 115, α-Asn 157 and α-Asn 295 β-Asn 84, β-Asn 142, β-Asn 190 and β-Asn 327. <scene name='Beta-Hexosaminidase/Opening/8'>Mutations</scene> in the α-subunit are associated with TS disease and with Late Onset Tay Sachs disease (LOTS) (Chronic & Acute clinical phenotype). Interestingly, <scene name='Beta-Hexosaminidase/Mut_2/2'>α-G269S</scene> is the most common mutation associated with LOTS disease.

===References===
<references/>
__NOEDITSECTION__

Beta-Hexosaminidase

2011-02-17T10:44:36Z

Harry Greenblatt:

<applet load="" size="600" color="" frame="true" spin="on" Scene ="Beta-Hexosaminidase/Opening/3" align="right" caption="Crystal Structure of Human β-Hexosaminidase, ([[2gjx]])"/>
===Structure of Human β-Hexosaminidase A and its association with Tay-Sachs disease===
<hr/>
β-Hexosaminidase A is a lysosomal enzyme essential for the degradation of GM2 gangliosides. Deficiency of lysosomal β-Hexosaminidase A due to inherited defects in the α-subunit gene results in Tay-Sachs (TS) disease. The 3D structure of β-Hexosaminidase A was determined by the group of Michael N.G. James at the University of Alberta, Edmonton, Canada.<ref>PMID:16698036</ref> The structure reveals an <scene name='Beta-Hexosaminidase/Subunits/4'>αβ-heterodimer</scene>, with each subunit having a functional active site. Only the <scene name='Beta-Hexosaminidase/Alpha/2'>α-subunit</scene> active site can hydrolyze GM2 gangliosides due to <scene name='Beta-Hexosaminidase/Gsep/6'>a flexible loop</scene> α280GSEP283 structure that is removed post-translationaly from β, and to the presence of <scene name='Beta-Hexosaminidase/4234/5'>α-Asn 423 and α-Arg 424</scene>. The <scene name='Beta-Hexosaminidase/Gsep_out/3'>loop structure</scene> is involved in binding the GM2 activator protein, while <scene name='Beta-Hexosaminidase/424_b/1'>α-Arg424</scene> is critical for binding the carboxylate group of the N-acetyl-neuraminic acid residue of GM2. <scene name='Beta-Hexosaminidase/2_active/1'>Two active sites</scene> are present in HexA dimer; one comprising residues from <scene name='Beta-Hexosaminidase/Active_alpha/1'>the α-subunit</scene> (R178 D207 H262 E323 D322 W373 W392 W460 Y421 R424 N423 E462) and a second one from residues of the <scene name='Beta-Hexosaminidase/Active_beta/1'>β-subunit</scene> (R211 D240 H294 E355 D354 W405 W424 Y450 L453 D452 E491 W489). These active sites are located at the opening of TIM barrels at the interface between the α and β-subunits. The HexA <scene name='Beta-Hexosaminidase/Glyco/1'>undergoes glycosylation</scene> on the α and β-subunits; α-Asn 115, α-Asn 157 and α-Asn 295 β-Asn 84, β-Asn 142, β-Asn 190 and β-Asn 327. <scene name='Beta-Hexosaminidase/Opening/8'>Mutations</scene> in the α-subunit are associated with TS disease and with Late Onset Tay Sachs disease (LOTS) (Chronic & Acute clinical phenotype). Interestingly, <scene name='Beta-Hexosaminidase/Mut_2/2'>α-G269S</scene> is the most common mutation associated with LOTS disease.

===References===
<references/>
__NOEDITSECTION__

Beta-Hexosaminidase

2011-02-17T10:42:12Z

Harry Greenblatt: Fix up small mistakes

<applet load="" size="600" color="" frame="true" spin="on" Scene ="Beta-Hexosaminidase/Opening/3" align="right" caption="Crystal Structure of Human β-Hexosaminidase, ([[2gjx]])"/>
===Structure of Human β-Hexosaminidase A and its association with Tay-Sachs disease===
<hr/>
β-Hexosaminidase A is a lysosomal enzyme essential for the degradation of GM2 gangliosides. Deficiency of lysosomal β-Hexosaminidase A due to inherited defects in the α-subunit gene results in Tay-Sachs (TS) disease. The 3D structure of β-Hexosaminidase A was determined by the group of Michael N.G. James at the University of Alberta, Edmonton, Canada.<ref>PMID:16698036</ref> The structure reveals an <scene name='Beta-Hexosaminidase/Subunits/4'>αβ-heterodimer</scene>, with each subunit having a functional active site. Only the <scene name='Beta-Hexosaminidase/Alpha/2'>α-subunit</scene> active site can hydrolyze GM2 gangliosides due to <scene name='Beta-Hexosaminidase/Gsep/6'>a flexible loop</scene> α280GSEP283 structure that is removed post-translationaly from β, and to the presence of <scene name='Beta-Hexosaminidase/4234/5'>α-Asn 423 and α-Arg 424</scene>. The <scene name='Beta-Hexosaminidase/Gsep_out/3'>loop structure</scene> is involved in binding the GM2 activator protein, while <scene name='Beta-Hexosaminidase/424_b/1'>α-Arg424</scene> is critical for binding the carboxylate group of the N-acetyl-neuraminic acid residue of GM2. <scene name='Beta-Hexosaminidase/2_active/1'>Two active sites</scene> are present in HexA dimer; one comprising residues from <scene name='Beta-Hexosaminidase/Active_alpha/1'>the α-subunit</scene> (R178 D207 H262 E323 D322 W373 W392 W460 Y421 R424 N423 E462) and a second one from residues of the <scene name='Beta-Hexosaminidase/Active_beta/1'>β-subunit</scene> (R211 D240 H294 E355 D354 W405 W424 Y450 L453 D452 E491 W489). These active sites are located at the opening of a TIM barrels at the interface between the α and β-subunits. The HexA <scene name='Beta-Hexosaminidase/Glyco/1'>undergoes glycosylation</scene> on the α and β-subunits; α-Asn 115, α-Asn 157 and α-Asn 295 β-Asn 84, β-Asn 142, β-Asn 190 and β-Asn 327. <scene name='Beta-Hexosaminidase/Opening/8'>Mutations</scene> in the α-subunit are associated with TS disease and with Late Onset Tay Sachs disease (LOTS) (Chronic & Acute clinical phenotype). Interestingly, <scene name='Beta-Hexosaminidase/Mut_2/2'>α-G269S</scene> is the most common mutation associated with LOTS disease.

===References===
<references/>
__NOEDITSECTION__

Beta-Hexosaminidase

2011-02-17T10:37:55Z

Harry Greenblatt:

<applet load="" size="600" color="" frame="true" spin="on" Scene ="Beta-Hexosaminidase/Opening/3" align="right" caption="Crystal Structure of Human β-Hexosaminidase, ([[2gjx]])"/>
===Structure of Human β-Hexosaminidase A and its association with Tay-Sachs disease===
<hr/>
β-Hexosaminidase A is a lysosomal enzyme essential for the degradation of GM2 gangliosides. Deficiency of lysosomal β-Hexosaminidase A due to inherited defects in the α-subunit gene results in Tay-Sachs (TS) disease. The 3D structure of β-Hexosaminidase A was determined by the group of Michael N.G. James at the University of Alberta, Edmonton, Canada.<ref>PMID:16698036</ref> The structure reveals an <scene name='Beta-Hexosaminidase/Subunits/4'>αβ-heterodimer</scene>, with each subunit having a functional active site. Only the <scene name='Beta-Hexosaminidase/Alpha/2'>α-subunit</scene> active site can hydrolyze GM2 gangliosides due to <scene name='Beta-Hexosaminidase/Gsep/6'>a flexible loop</scene> α280GSEP283 structure that is removed post-translationaly from β, and to the presence of <scene name='Beta-Hexosaminidase/4234/5'>α-Asn 423 and α-Arg 424</scene>. The <scene name='Beta-Hexosaminidase/Gsep_out/3'>loop structure</scene> is involved in binding the GM2 activator protein, while <scene name='Beta-Hexosaminidase/424_b/1'>α-Arg424</scene> is critical for binding the carboxylate group of the N-acetyl-neuraminic acid residue of GM2. <scene name='Beta-Hexosaminidase/2_active/1'>Two active sites</scene> are present in HexA dimer; one comprising residues from <scene name='Beta-Hexosaminidase/Active_alpha/1'>the α-subunit</scene> (R178 D207 H262 E323 D322 W373 W392 W460 Y421 R424 N423 E462) and a second one from residues of the <scene name='Beta-Hexosaminidase/Active_beta/1'>β-subunit</scene> (R211 D240 H294 E355 D354 W405 W424 Y450 L453 D452 E491 W489). These active sites are located at the opening of a TIM barrels at the interface between the α and β-subunits. The HexA <scene name='Beta-Hexosaminidase/Glyco/1'>undergoes glycosylation</scene> at the α and β-subunits; α-Asn 115, α-Asn 157 and α-Asn 295 β-Asn 84, β-Asn 142, β-Asn 190 and β-Asn 327. <scene name='Beta-Hexosaminidase/Opening/8'>Mutations</scene> in the α-subunit are associated with TS disease and with Late Onset Tay Sachs disease (LOTS) (Chronic & Acute clinical phenotype). Interestingly, <scene name='Beta-Hexosaminidase/Mut_2/2'>α-G269S</scene> is the most common mutation associated with LOTS disease.

===References===
<references/>
__NOEDITSECTION__

Beta-Hexosaminidase

2011-02-17T10:35:25Z

Harry Greenblatt:

<applet load="" size="600" color="" frame="true" spin="on" Scene ="Beta-Hexosaminidase/Opening/3" align="right" caption="Crystal Structure of Human β-Hexosaminidase, ([[2gjx]])"/>
===Structure of Human β-Hexosaminidase A and its association with Tay-Sachs disease===
<hr/>
β-Hexosaminidase A is a lysosomal enzyme essential for the degradation of GM2 gangliosides. Deficiency of lysosomal β-Hexosaminidase A due to inherited defects in the α-subunit gene results in Tay-Sachs (TS) disease. The 3D structure of β-Hexosaminidase A was determined by the group of Michael N.G. James at the University of Alberta, Edmonton, Canada.<ref>PMID:16698036</ref> The structure reveals an <scene name='Beta-Hexosaminidase/Subunits/4'>αβ-heterodimer</scene>, with each subunit having a functional active site. Only the <scene name='Beta-Hexosaminidase/Alpha/2'>α-subunit</scene> active site can hydrolyze GM2 gangliosides due to <scene name='Beta-Hexosaminidase/Gsep/6'>a flexible loop</scene> α280GSEP283 structure that is removed post-translational from β, and to the presence of <scene name='Beta-Hexosaminidase/4234/5'>α-Asn 423 and α-Arg 424</scene>. The <scene name='Beta-Hexosaminidase/Gsep_out/3'>loop structure</scene> is involved in binding the GM2 activator protein, while <scene name='Beta-Hexosaminidase/424_b/1'>α-Arg424</scene> is critical for binding the carboxylate group of the N-acetyl-neuraminic acid residue of GM2. <scene name='Beta-Hexosaminidase/2_active/1'>Two active sites</scene> are present in HexA dimmer; one comprising residues from <scene name='Beta-Hexosaminidase/Active_alpha/1'>the α-subunit</scene> (R178 D207 H262 E323 D322 W373 W392 W460 Y421 R424 N423 E462) and a second one from residues of the <scene name='Beta-Hexosaminidase/Active_beta/1'>β-subunit</scene> (R211 D240 H294 E355 D354 W405 W424 Y450 L453 D452 E491 W489). These active sites are located at the opening of a TIM barrels at the interface between the α and β-subunits. The HexA <scene name='Beta-Hexosaminidase/Glyco/1'>undergoes glycosylation</scene> at the α and β-subunits; α-Asn 115, α-Asn 157 and α-Asn 295 β-Asn 84, β-Asn 142, β-Asn 190 and β-Asn 327. <scene name='Beta-Hexosaminidase/Opening/8'>Mutations</scene> in the a-subunit are associated with TS disease and with Late Onset Tay Sachs disease (LOTS) (Chronic & Acute clinical phenotype). Interestingly, <scene name='Beta-Hexosaminidase/Mut_2/2'>α-G269S</scene> is the most common mutation associated with LOTS disease.

===References===
<references/>
__NOEDITSECTION__

File:HuBChE.png

2010-04-20T12:50:58Z

Harry Greenblatt: Human Butyrylcholinesterase from 1p0i looking down the gorge, showing Trp82, and the catalytic triad (Ser 198, His 438, Glu 325) as spheres (PyMOL).

== Summary ==
Human Butyrylcholinesterase from 1p0i looking down the gorge, showing Trp82, and the catalytic triad (Ser 198, His 438, Glu 325) as spheres (PyMOL).
== Licensing ==
{{self|cc-by-3.0}}

File:1w76 ss.pdb

2009-06-23T06:53:11Z

Harry Greenblatt: In the original PDB entry, secondary structure definitions exist only for Chain A, but not for Chain B. This has been added here.

In the original PDB entry, secondary structure definitions exist only for Chain A, but not for Chain B. This has been added here.

File:1W4Lnew.png

2009-06-21T12:06:33Z

Harry Greenblatt: Bis acting Galanthamine derivative (yellow carbon atoms) bound to TcAChE, showing Asp72, Trp84, Ser200, Trp279, and His440 (green carbon atoms). See PDB file 1W4L.

== Summary ==
Bis acting Galanthamine derivative (yellow carbon atoms) bound to TcAChE, showing Asp72, Trp84, Ser200, Trp279, and His440 (green carbon atoms).
See PDB file 1W4L.
== Licensing ==
{{self|cc-by-3.0}}

File:1and3.png

2009-06-21T11:42:06Z

Harry Greenblatt: /* Summary */

== Summary ==
Native galanthamine (1) and a bis-acting iminium phthalimido derivative (3)

== Licensing ==
{{self|cc-by-3.0}}

File:1and3.png

2009-06-21T11:41:38Z

Harry Greenblatt: Native galanthamine (1) and a bis-acting iminium phthalimido derivative (3)

== Summary ==
Native galanthamine (1) and a bis-acting iminium phthalimido derivative (3)
== Licensing ==
{{self|cc-by-3.0}}

File:1W6R new.png

2009-06-21T09:12:42Z

Harry Greenblatt: Pymol generated image showing Galanthamine derivative (yellow carbon atoms) bound in active site of TcAChE. Enzyme residues Asp72, Trp84, Ser200, Trp279, and His440 shown with green carbon atoms.

== Summary ==
Pymol generated image showing Galanthamine derivative (yellow carbon atoms) bound in active site of TcAChE. Enzyme residues Asp72, Trp84, Ser200, Trp279, and His440 shown with green carbon atoms.
== Licensing ==
{{self|cc-by-3.0}}

File:1and5.png

2009-06-18T07:01:22Z

Harry Greenblatt:

Native Galanthamine (1) and the iminium derivative (5) used to form the complex found in [[1w6r]]

File:1and5.png

2009-06-18T06:43:15Z

Harry Greenblatt:

Native Galanthamine (1) and the imine derivative (5) used to form the complex found in [[1w6r]]

File:1and5.png

2009-06-18T06:42:03Z

Harry Greenblatt: Native Galanthamine (1) and the imine derivative (5) used to form this complex

Native Galanthamine (1) and the imine derivative (5) used to form this complex

File:Sgap1.png

2008-10-02T13:13:30Z

Harry Greenblatt: /* Summary */

== Summary ==
PCI-1 bound in the active site of SGPB, showing the surface of the enzyme that interacts with the inhibitor. The surface generated by His57 and Ser195 of the catalytic triad are coloured blue and raspberry, respectively. The surface of the disulphide bond between Cys42 and Cys58 is coloured yellow.

== Licensing ==
{{self|cc-by-3.0}}

File:Sgap1.png

2008-10-02T13:06:00Z

Harry Greenblatt: View showing PCI-1 bound in the active site of SGPB showing surface of interaction on enzyme

== Summary ==
View showing PCI-1 bound in the active site of SGPB showing surface of interaction on enzyme
== Licensing ==
{{self|cc-by-3.0}}

Serine endoproteinase

2008-09-22T07:33:07Z

Harry Greenblatt: Redirecting to Serine Protease

#REDIRECT [[Serine Protease]]

Serine endoproteinase

2008-09-22T07:30:49Z

Harry Greenblatt: Redirecting to Serine protease

#REDIRECT [[serine protease]]

Streptomyces griseus Aminopeptidase (SGAP)

2008-09-22T07:22:08Z

Harry Greenblatt: /* Historical Context */

[[Image:1xjo_cartoon.png | 330px | thumb | S griseus aminopeptidase, showing overall fold. Zinc ions are dark grey, calcium ion is white.]]
'''Title''' : Structure of ''S. griseus'' aminopeptidase

== ''Streptomyces griseus'' Aminopeptidase (SGAP) ==

==Biological function==
''S. griseus'' Aminopeptidase (SGAP; E.C. 3.4.11.-) cleaves the N-terminal amino acid from a peptide or protein, and is specific for larger hydrophobic acids, especially leucine. No cleavage occurs if the next residue is proline.

[[Image:aminopeptidase_rxn2.png | left | thumb| 800px | Reaction catalyzed by SGAP; scissile bond is shown in red.]]
 

== Biological Context ==

SGAP is one of the many proteinases present in the extracellular fluid of cultures of ''Streptomyces griseus'', and can be isolated from [http://en.wikipedia.org/wiki/Pronase Pronase], the commercial preparation of the extracellular fluid from this organism. SGAP is a monomeric, 30KDa, heat stable enzyme requiring two Zn2+ ions for activity, and is activated by Ca2+.

== Structures Available ==

* [[1xjo]] - native SGAP to 1.75Å
* [[1cp7]] - native SGAP to 1.58Å
* [[1qq9]] - SGAP complexed with L-methionine to 1.53Å
* [[1f2o]] - complex with L-leucine to 1.70Å
* [[1f2p]] - complex with L-phenylalanine to 1.80Å
* [[1tf8]] - complex with L-tryptophane to 1.30Å
* [[1tf9]] - complex with ''p''-iodo-L-phenylalanine to 1.30Å
* [[1tkf]] - with D-tryptophan to 1.20Å
* [[1tkh]] - with D-phenylalanine to 1.25Å
* [[1tkj]] - with D-methionine to 1.15Å
* [[1xbu]] - with ''p''-iodo-D-phenylalanine to 1.20Å

== Historical Context ==

The proteolytic activity contained in the extracellular fluid of cultures of ''Streptomyces griseus'' was first identified by Nomoto and Narahashi (1959a), who obtained a highly purified preparation of this activity from the K-1 strain of this bactreria. A large scale version of their procedure was used to prepare commercial quantities of this preparation (Pronase). Various physical criteria showed that Pronase was homogeneous (Nomoto and Narahashi, 1959b), yet displayed both exopeptidase and endopeptidase activity, with a wide range of side chain specificities (Nomoto and Narahashi, 1959b, 1959c; Nomoto ''et al''., 1960a, 1960b, 1960c). The supposed homogeneity of Pronase was controversial, with other investigators using various chromatographic methods to isolate more fractions with proteolytic activity (Hiramatsu and Ouchi, 1963; Nomoto et al., 1964). Subsequently Narahashi and Yanagita (1967) identified several distinct proteolytic activities including one which had aminopeptidase activity, and was activated by Ca2+ and Co2+. In contrast to other proteinase activities in the mixture, this aminopeptidase activity displayed considerable heat stablity (up to 80℃) and was unaffected by 9M urea. The activity, was, however, very sensitive to metal chelating agents.

While attempting to isolate the protein responsible for the trypsin activity in Pronase, Vosbeck ''et al.'' (1973) isolated two fractions with aminopeptidase activity. Although the two fractions differed slighty in their molecular weights (23K and 25K), they appeared to have the same enzymatic properties.

Interest in SGAP was renewed when aminopeptidases were recognized as useful tools in assays of metalloendopeptidase activity. The assay was based on a two stage reaction, with the endopeptidase cleaved an N-blocked peptide to release smaller peptide that was rapidly degraded by an aminopeptidase, generating a chromophore (Orlowski and Wilk, 1981; Mumford ''et al''., 1981). SGAP was considered an ideal tool for this purpose, given its stability, small size, and availability (Indig ''et al''., 1990).

== References ==


* Hiramatsu, A., & Ouchi, T. (1963). On the proteolytic enzymes from the commercial protease preparation of ''Streptomyces griseus'' (Pronase P). ''J. Biochem''. '''54''', 462-464.
* Indig, F.E., Benayahu, D., Fried, A., Wientroub, S., Blumberg, S. (1990). Neutral endopeptidase (EC 3.4.24.11) is highly expressed on osteoblastic cells and other marrow stromal cell types. ''Biochem. Biophys. Res. Commun''. '''172''', 620-626. [http://www.ncbi.nlm.nih.gov/pubmed/2241957?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 2241957]
* Mumford, R.A., Pierzchala, P.A., Strauss, A.W., Zimmerman, M. (1981). Purification of a membrane-bound metalloendopeptidase from porcine kidney that degrades peptide hormones. ''Proc. Natl Acad. Sci. USA'' '''78''', 6623-6627. [http://www.ncbi.nlm.nih.gov/pubmed/7031658?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7031658]
* Narahashi, Y., & Yanagita, M. (1967). Studies on proteolytic enzymes (Pronase) of S''treptomyces griseus'' K-1. I. Nature and properties of the proteolytic enzyme system. ''J. Biochem''. (Tokyo) '''62''', 633-641. [http://www.ncbi.nlm.nih.gov/pubmed/4968616?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4968616 ]
* Nomoto, M., & Narahashi, Y. (1959a). A proteolytic enzyme of ''Streptomyces griseus'': I. Purification of a protease of ''Streptomyces grisues''. ''J. Biochem.'' '''46''', 653-667.
* Nomoto, M., & Narahashi, Y. (1959b). A proteolytic enzyme of ''Streptomyces griseus'': III. Homogeneity of the purified enzyme preparation. ''J. Biochem.'' '''46''', 1481-1487.
* Nomoto, M., & Narahashi, Y. (1959c). A proteolytic enzyme of ''Streptomyces griseus'': IV. General properties of ''Streptomyces grisues'' protease. ''J. Biochem.'' '''46''', 1645-1651.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960a). A proteolytic enzyme of ''Streptomyces griseus'': V. Protective effect of calcium ion on the stability of protease. ''J. Biochem.'' '''48''', 453-463.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960b). A proteolytic enzyme of ''Streptomyces griseus'': VI. Hydrolysis of protein by ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 593-602.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960c). A proteolytic enzyme of ''Streptomyces griseus'': VII. Substrate specificity of ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 906-918.
* Nomoto, M., Narahashi, Y., Ouchi, T., & Hiramatsu, A. (1964). Abstract, 6th Inern. Congr. Biochem., N.Y., '''4''', 123.
* Orlowski, M., & Wilk, S. (1981). Purification and specificity of a membrane-bound metalloendopeptidase from bovine pituitaries. ''Biochemistry'' '''20''', 4942-4950. [http://www.ncbi.nlm.nih.gov/pubmed/7028098?ordinalpos=12&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7028098]
* Vosbeck, K. D., Chow, K. F., & Awad, W. M. Jr (1973). The proteolytic enzymes of the K-1 strain of ''Streptomyces griseus'' obtained from a commercial preparation (Pronase). Purification and characterization of the aminopeptidases. '' J. Biol. Chem''. '''248''', 6029-6034. [http://www.ncbi.nlm.nih.gov/pubmed/4199257?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4199257]

--[[User:Harry|Harry]] 15:20, 17 March 2008 (IST)

Streptomyces griseus Aminopeptidase (SGAP)

2008-09-22T07:21:31Z

Harry Greenblatt: /* Historical Context */

[[Image:1xjo_cartoon.png | 330px | thumb | S griseus aminopeptidase, showing overall fold. Zinc ions are dark grey, calcium ion is white.]]
'''Title''' : Structure of ''S. griseus'' aminopeptidase

== ''Streptomyces griseus'' Aminopeptidase (SGAP) ==

==Biological function==
''S. griseus'' Aminopeptidase (SGAP; E.C. 3.4.11.-) cleaves the N-terminal amino acid from a peptide or protein, and is specific for larger hydrophobic acids, especially leucine. No cleavage occurs if the next residue is proline.

[[Image:aminopeptidase_rxn2.png | left | thumb| 800px | Reaction catalyzed by SGAP; scissile bond is shown in red.]]
 

== Biological Context ==

SGAP is one of the many proteinases present in the extracellular fluid of cultures of ''Streptomyces griseus'', and can be isolated from [http://en.wikipedia.org/wiki/Pronase Pronase], the commercial preparation of the extracellular fluid from this organism. SGAP is a monomeric, 30KDa, heat stable enzyme requiring two Zn2+ ions for activity, and is activated by Ca2+.

== Structures Available ==

* [[1xjo]] - native SGAP to 1.75Å
* [[1cp7]] - native SGAP to 1.58Å
* [[1qq9]] - SGAP complexed with L-methionine to 1.53Å
* [[1f2o]] - complex with L-leucine to 1.70Å
* [[1f2p]] - complex with L-phenylalanine to 1.80Å
* [[1tf8]] - complex with L-tryptophane to 1.30Å
* [[1tf9]] - complex with ''p''-iodo-L-phenylalanine to 1.30Å
* [[1tkf]] - with D-tryptophan to 1.20Å
* [[1tkh]] - with D-phenylalanine to 1.25Å
* [[1tkj]] - with D-methionine to 1.15Å
* [[1xbu]] - with ''p''-iodo-D-phenylalanine to 1.20Å

== Historical Context ==

The proteolytic activity contained in the extracellular fluid of cultures of ''Streptomyces griseus'' was first identified by Nomoto and Narahashi (1959a), who obtained a highly purified preparation of this activity from the K-1 strain of this bactreria. A large scale version of their procedure was used to prepare commercial quantities of this preparation (Pronase). Various physical criteria showed that Pronase was homogeneous (Nomoto and Narahashi, 1959b), yet displayed both exopeptidase and endopeptidase activity, with a wide range of side chain specificities (Nomoto and Narahashi, 1959b, 1959c; Nomoto ''et al''., 1960a; 1960b; 1960c). The supposed homogeneity of Pronase was controversial, with other investigators using various chromatographic methods to isolate more fractions with proteolytic activity (Hiramatsu and Ouchi, 1963; Nomoto et al., 1964). Subsequently Narahashi and Yanagita (1967) identified several distinct proteolytic activities including one which had aminopeptidase activity, and was activated by Ca2+ and Co2+. In contrast to other proteinase activities in the mixture, this aminopeptidase activity displayed considerable heat stablity (up to 80℃) and was unaffected by 9M urea. The activity, was, however, very sensitive to metal chelating agents.

While attempting to isolate the protein responsible for the trypsin activity in Pronase, Vosbeck ''et al.'' (1973) isolated two fractions with aminopeptidase activity. Although the two fractions differed slighty in their molecular weights (23K and 25K), they appeared to have the same enzymatic properties.

Interest in SGAP was renewed when aminopeptidases were recognized as useful tools in assays of metalloendopeptidase activity. The assay was based on a two stage reaction, with the endopeptidase cleaved an N-blocked peptide to release smaller peptide that was rapidly degraded by an aminopeptidase, generating a chromophore (Orlowski and Wilk, 1981; Mumford ''et al''., 1981). SGAP was considered an ideal tool for this purpose, given its stability, small size, and availability (Indig ''et al''., 1990).

== References ==


* Hiramatsu, A., & Ouchi, T. (1963). On the proteolytic enzymes from the commercial protease preparation of ''Streptomyces griseus'' (Pronase P). ''J. Biochem''. '''54''', 462-464.
* Indig, F.E., Benayahu, D., Fried, A., Wientroub, S., Blumberg, S. (1990). Neutral endopeptidase (EC 3.4.24.11) is highly expressed on osteoblastic cells and other marrow stromal cell types. ''Biochem. Biophys. Res. Commun''. '''172''', 620-626. [http://www.ncbi.nlm.nih.gov/pubmed/2241957?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 2241957]
* Mumford, R.A., Pierzchala, P.A., Strauss, A.W., Zimmerman, M. (1981). Purification of a membrane-bound metalloendopeptidase from porcine kidney that degrades peptide hormones. ''Proc. Natl Acad. Sci. USA'' '''78''', 6623-6627. [http://www.ncbi.nlm.nih.gov/pubmed/7031658?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7031658]
* Narahashi, Y., & Yanagita, M. (1967). Studies on proteolytic enzymes (Pronase) of S''treptomyces griseus'' K-1. I. Nature and properties of the proteolytic enzyme system. ''J. Biochem''. (Tokyo) '''62''', 633-641. [http://www.ncbi.nlm.nih.gov/pubmed/4968616?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4968616 ]
* Nomoto, M., & Narahashi, Y. (1959a). A proteolytic enzyme of ''Streptomyces griseus'': I. Purification of a protease of ''Streptomyces grisues''. ''J. Biochem.'' '''46''', 653-667.
* Nomoto, M., & Narahashi, Y. (1959b). A proteolytic enzyme of ''Streptomyces griseus'': III. Homogeneity of the purified enzyme preparation. ''J. Biochem.'' '''46''', 1481-1487.
* Nomoto, M., & Narahashi, Y. (1959c). A proteolytic enzyme of ''Streptomyces griseus'': IV. General properties of ''Streptomyces grisues'' protease. ''J. Biochem.'' '''46''', 1645-1651.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960a). A proteolytic enzyme of ''Streptomyces griseus'': V. Protective effect of calcium ion on the stability of protease. ''J. Biochem.'' '''48''', 453-463.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960b). A proteolytic enzyme of ''Streptomyces griseus'': VI. Hydrolysis of protein by ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 593-602.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960c). A proteolytic enzyme of ''Streptomyces griseus'': VII. Substrate specificity of ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 906-918.
* Nomoto, M., Narahashi, Y., Ouchi, T., & Hiramatsu, A. (1964). Abstract, 6th Inern. Congr. Biochem., N.Y., '''4''', 123.
* Orlowski, M., & Wilk, S. (1981). Purification and specificity of a membrane-bound metalloendopeptidase from bovine pituitaries. ''Biochemistry'' '''20''', 4942-4950. [http://www.ncbi.nlm.nih.gov/pubmed/7028098?ordinalpos=12&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7028098]
* Vosbeck, K. D., Chow, K. F., & Awad, W. M. Jr (1973). The proteolytic enzymes of the K-1 strain of ''Streptomyces griseus'' obtained from a commercial preparation (Pronase). Purification and characterization of the aminopeptidases. '' J. Biol. Chem''. '''248''', 6029-6034. [http://www.ncbi.nlm.nih.gov/pubmed/4199257?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4199257]

--[[User:Harry|Harry]] 15:20, 17 March 2008 (IST)

Serine protease

2008-09-22T07:16:57Z

Harry Greenblatt:

<applet load="1ppb" size="350" color="white" frame="true" align="left" spinBox="true"
caption="Human Thrombin with PPACK inhibitor" />

'''Serine proteases''', or '''proteinases''', so called due to the presence of a serine residue in the active site, are a class of enzymes that catalyse the hydrolysis of peptide bonds in proteins.

==Thrombin==
'''Thrombin''' is a "trypsin-like" serine protease. Its structure (PDB code [[1ppb]]) is shown here with a peptide chloroketone inhibitor (PPACK). The thrombin A chain (cleaved N terminal fragement) is shown in cyan and the B chain is shown in red. The <scene name='Serine_Protease/Active_site/2'>Active site</scene> is made up of a catalytic triad of Ser195, His57 and Asp102, backed up by Ser214. The peptide chloroketone inhibitor (PPACK) is shown in purple. A closeup shows the <scene name='Serine_Protease/Activation_site/2'>activation site</scene> at which the sidechain of Asp194 makes a salt link with the N-terminus at residue 16, newly formed when the A chain is cleaved in the zymogen-to-enzyme activation process. The specificity pocket is on one side of the throat of the domain 2 beta barrel, and the activation site is close next to it.

The B chain consists of <scene name='Serine_Protease/Domains/1'>two domains</scene>. As is true for all of the "trypsin-like" serine proteases, each of the two thrombin domains consists mainly of a 6-stranded, antiparallel beta barrel. The specificity pocket (here filled with the Lys sidechain of the PPACK inhibitor) is in one side of the throat of the domain 2beta barrel, and the activation site is close next to it.

<applet load="2ptc" size="350" color="white" frame="true" align="right" spinBox="true"
caption="Trypsin BPT1 complex" />

==Trypsin-BPTI complex==
The trypsin backbone is shown in pink and the trypsin inhibitor, BPTI, in yellow (PDB code [[2ptc]]). The <scene name='Serine_Protease/Active_site/3'>active site</scene> residues [Ser195-His57-Asp102-Ser214] are shown in green, the disulfide bond between residues 14-38 is shown in yellow and the Lys 15 sidechain at the specificity site in pink.

{{Clear}}
== '''Content Donators''' ==

* Content for this page has been included and adapted with permission from Jane S. and David C. Richardson's http://kinemage.biochem.duke.edu/

Serine protease

2008-09-22T07:15:46Z

Harry Greenblatt: correct spelling mistakes

<applet load="1ppb" size="350" color="white" frame="true" align="left" spinBox="true"
caption="Human Thrombin with PPACK inhibitor" />

'''Serine proteases''', so called due to the presence of a serine residue in the active site, are a class of enzymes that catalyse the hydrolysis of peptide bonds in proteins.

==Thrombin==
'''Thrombin''' is a "trypsin-like" serine protease. Its structure (PDB code [[1ppb]]) is shown here with a peptide chloroketone inhibitor (PPACK). The thrombin A chain (cleaved N terminal fragement) is shown in cyan and the B chain is shown in red. The <scene name='Serine_Protease/Active_site/2'>Active site</scene> is made up of a catalytic triad of Ser195, His57 and Asp102, backed up by Ser214. The peptide chloroketone inhibitor (PPACK) is shown in purple. A closeup shows the <scene name='Serine_Protease/Activation_site/2'>activation site</scene> at which the sidechain of Asp194 makes a salt link with the N-terminus at residue 16, newly formed when the A chain is cleaved in the zymogen-to-enzyme activation process. The specificity pocket is on one side of the throat of the domain 2 beta barrel, and the activation site is close next to it.

The B chain consists of <scene name='Serine_Protease/Domains/1'>two domains</scene>. As is true for all of the "trypsin-like" serine proteases, each of the two thrombin domains consists mainly of a 6-stranded, antiparallel beta barrel. The specificity pocket (here filled with the Lys sidechain of the PPACK inhibitor) is in one side of the throat of the domain 2beta barrel, and the activation site is close next to it.

<applet load="2ptc" size="350" color="white" frame="true" align="right" spinBox="true"
caption="Trypsin BPT1 complex" />

==Trypsin-BPTI complex==
The trypsin backbone is shown in pink and the trypsin inhibitor, BPTI, in yellow (PDB code [[2ptc]]). The <scene name='Serine_Protease/Active_site/3'>active site</scene> residues [Ser195-His57-Asp102-Ser214] are shown in green, the disulfide bond between residues 14-38 is shown in yellow and the Lys 15 sidechain at the specificity site in pink.

{{Clear}}
== '''Content Donators''' ==

* Content for this page has been included and adapted with permission from Jane S. and David C. Richardson's http://kinemage.biochem.duke.edu/

Streptomyces griseus Aminopeptidase (SGAP)

2008-09-22T07:05:48Z

Harry Greenblatt: /* Biological Context */

[[Image:1xjo_cartoon.png | 330px | thumb | S griseus aminopeptidase, showing overall fold. Zinc ions are dark grey, calcium ion is white.]]
'''Title''' : Structure of ''S. griseus'' aminopeptidase

== ''Streptomyces griseus'' Aminopeptidase (SGAP) ==

==Biological function==
''S. griseus'' Aminopeptidase (SGAP; E.C. 3.4.11.-) cleaves the N-terminal amino acid from a peptide or protein, and is specific for larger hydrophobic acids, especially leucine. No cleavage occurs if the next residue is proline.

[[Image:aminopeptidase_rxn2.png | left | thumb| 800px | Reaction catalyzed by SGAP; scissile bond is shown in red.]]
 

== Biological Context ==

SGAP is one of the many proteinases present in the extracellular fluid of cultures of ''Streptomyces griseus'', and can be isolated from [http://en.wikipedia.org/wiki/Pronase Pronase], the commercial preparation of the extracellular fluid from this organism. SGAP is a monomeric, 30KDa, heat stable enzyme requiring two Zn2+ ions for activity, and is activated by Ca2+.

== Structures Available ==

* [[1xjo]] - native SGAP to 1.75Å
* [[1cp7]] - native SGAP to 1.58Å
* [[1qq9]] - SGAP complexed with L-methionine to 1.53Å
* [[1f2o]] - complex with L-leucine to 1.70Å
* [[1f2p]] - complex with L-phenylalanine to 1.80Å
* [[1tf8]] - complex with L-tryptophane to 1.30Å
* [[1tf9]] - complex with ''p''-iodo-L-phenylalanine to 1.30Å
* [[1tkf]] - with D-tryptophan to 1.20Å
* [[1tkh]] - with D-phenylalanine to 1.25Å
* [[1tkj]] - with D-methionine to 1.15Å
* [[1xbu]] - with ''p''-iodo-D-phenylalanine to 1.20Å

== Historical Context ==

The proteolytic activity contained in the extracellular fluid of cultures of ''Streptomyces griseus'' was first identified by Nomoto and Narahashi (1959a), who obtained a highly purified preparation of this activity from the K-1 strain of this bactreria. A large scale version of their procedure was used to prepare commercial quantities of this preparation (Pronase). Various physical criteria showed that Pronase was homogeneous (Nomoto and Narahashi, 1959b), yet displayed both exopeptidase and endopeptidase activity, with a wide range of side chain specificities (Nomoto and Narahashi, 1959b, 1959c; Nomoto ''et al''., 1960a, 1960b; 1960c). The supposed homogeneity of Pronase was controversial, with other investigators using various chromatographic methods to isolate more fractions with proteolytic activity (Hiramatsu and Ouchi, 1963,; Nomoto et al., 1964). Subsequently Narahashi and Yanagita (1967) identified several distinct proteolytic activities including one which had aminopeptidase activity, and was activated by Ca2+ and Co2+. In contrast to other proteinase activities in the mixture, this aminopeptidase activity displayed considerable heat stablity (up to 80℃) and was unaffected by 9M urea. The activity, was, however, very sensitive to metal chelating agents.

While attempting to isolate the protein responsible for the trypsin activity in Pronase, Vosbeck ''et al.'' (1973) isolated two fractions with aminopeptidase activity. Although the two fractions differed slighty in their molecular weights (23K and 25K), they appeared to have the same enzymatic properties.

Interest in SGAP was renewed when aminopeptidases were recognized as useful tools in assays of metalloendopeptidase activity. The assay was based on a two stage reaction, with the endopeptidase cleaved an N-blocked peptide to release smaller peptide that was rapidly degraded by an aminopeptidase, generating a chromophore (Orlowski and Wilk, 1981; Mumford ''et al''., 1981). SGAP was considered an ideal tool for this purpose, given its stability, small size, and availability (Indig ''et al''., 1990).

== References ==


* Hiramatsu, A., & Ouchi, T. (1963). On the proteolytic enzymes from the commercial protease preparation of ''Streptomyces griseus'' (Pronase P). ''J. Biochem''. '''54''', 462-464.
* Indig, F.E., Benayahu, D., Fried, A., Wientroub, S., Blumberg, S. (1990). Neutral endopeptidase (EC 3.4.24.11) is highly expressed on osteoblastic cells and other marrow stromal cell types. ''Biochem. Biophys. Res. Commun''. '''172''', 620-626. [http://www.ncbi.nlm.nih.gov/pubmed/2241957?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 2241957]
* Mumford, R.A., Pierzchala, P.A., Strauss, A.W., Zimmerman, M. (1981). Purification of a membrane-bound metalloendopeptidase from porcine kidney that degrades peptide hormones. ''Proc. Natl Acad. Sci. USA'' '''78''', 6623-6627. [http://www.ncbi.nlm.nih.gov/pubmed/7031658?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7031658]
* Narahashi, Y., & Yanagita, M. (1967). Studies on proteolytic enzymes (Pronase) of S''treptomyces griseus'' K-1. I. Nature and properties of the proteolytic enzyme system. ''J. Biochem''. (Tokyo) '''62''', 633-641. [http://www.ncbi.nlm.nih.gov/pubmed/4968616?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4968616 ]
* Nomoto, M., & Narahashi, Y. (1959a). A proteolytic enzyme of ''Streptomyces griseus'': I. Purification of a protease of ''Streptomyces grisues''. ''J. Biochem.'' '''46''', 653-667.
* Nomoto, M., & Narahashi, Y. (1959b). A proteolytic enzyme of ''Streptomyces griseus'': III. Homogeneity of the purified enzyme preparation. ''J. Biochem.'' '''46''', 1481-1487.
* Nomoto, M., & Narahashi, Y. (1959c). A proteolytic enzyme of ''Streptomyces griseus'': IV. General properties of ''Streptomyces grisues'' protease. ''J. Biochem.'' '''46''', 1645-1651.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960a). A proteolytic enzyme of ''Streptomyces griseus'': V. Protective effect of calcium ion on the stability of protease. ''J. Biochem.'' '''48''', 453-463.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960b). A proteolytic enzyme of ''Streptomyces griseus'': VI. Hydrolysis of protein by ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 593-602.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960c). A proteolytic enzyme of ''Streptomyces griseus'': VII. Substrate specificity of ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 906-918.
* Nomoto, M., Narahashi, Y., Ouchi, T., & Hiramatsu, A. (1964). Abstract, 6th Inern. Congr. Biochem., N.Y., '''4''', 123.
* Orlowski, M., & Wilk, S. (1981). Purification and specificity of a membrane-bound metalloendopeptidase from bovine pituitaries. ''Biochemistry'' '''20''', 4942-4950. [http://www.ncbi.nlm.nih.gov/pubmed/7028098?ordinalpos=12&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7028098]
* Vosbeck, K. D., Chow, K. F., & Awad, W. M. Jr (1973). The proteolytic enzymes of the K-1 strain of ''Streptomyces griseus'' obtained from a commercial preparation (Pronase). Purification and characterization of the aminopeptidases. '' J. Biol. Chem''. '''248''', 6029-6034. [http://www.ncbi.nlm.nih.gov/pubmed/4199257?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4199257]

--[[User:Harry|Harry]] 15:20, 17 March 2008 (IST)

Streptomyces griseus Aminopeptidase (SGAP)

2008-09-22T07:01:06Z

Harry Greenblatt:

[[Image:1xjo_cartoon.png | 330px | thumb | S griseus aminopeptidase, showing overall fold. Zinc ions are dark grey, calcium ion is white.]]
'''Title''' : Structure of ''S. griseus'' aminopeptidase

== ''Streptomyces griseus'' Aminopeptidase (SGAP) ==

==Biological function==
''S. griseus'' Aminopeptidase (SGAP; E.C. 3.4.11.-) cleaves the N-terminal amino acid from a peptide or protein, and is specific for larger hydrophobic acids, especially leucine. No cleavage occurs if the next residue is proline.

[[Image:aminopeptidase_rxn2.png | left | thumb| 800px | Reaction catalyzed by SGAP; scissile bond is shown in red.]]
 

== Biological Context ==

SGAP is one of the many proteinases present in the extracellular fluid of cultures of "Streptomyces griseus", and can be isolated from [http://en.wikipedia.org/wiki/Pronase Pronase], the commercial preparation of the extracellular fluid from this organism. SGAP is a monomeric, 30KDa, heat stable enzyme requiring two Zn2+ ions for activity, and is activated by Ca2+.

== Structures Available ==

* [[1xjo]] - native SGAP to 1.75Å
* [[1cp7]] - native SGAP to 1.58Å
* [[1qq9]] - SGAP complexed with L-methionine to 1.53Å
* [[1f2o]] - complex with L-leucine to 1.70Å
* [[1f2p]] - complex with L-phenylalanine to 1.80Å
* [[1tf8]] - complex with L-tryptophane to 1.30Å
* [[1tf9]] - complex with ''p''-iodo-L-phenylalanine to 1.30Å
* [[1tkf]] - with D-tryptophan to 1.20Å
* [[1tkh]] - with D-phenylalanine to 1.25Å
* [[1tkj]] - with D-methionine to 1.15Å
* [[1xbu]] - with ''p''-iodo-D-phenylalanine to 1.20Å

== Historical Context ==

The proteolytic activity contained in the extracellular fluid of cultures of ''Streptomyces griseus'' was first identified by Nomoto and Narahashi (1959a), who obtained a highly purified preparation of this activity from the K-1 strain of this bactreria. A large scale version of their procedure was used to prepare commercial quantities of this preparation (Pronase). Various physical criteria showed that Pronase was homogeneous (Nomoto and Narahashi, 1959b), yet displayed both exopeptidase and endopeptidase activity, with a wide range of side chain specificities (Nomoto and Narahashi, 1959b, 1959c; Nomoto ''et al''., 1960a, 1960b; 1960c). The supposed homogeneity of Pronase was controversial, with other investigators using various chromatographic methods to isolate more fractions with proteolytic activity (Hiramatsu and Ouchi, 1963,; Nomoto et al., 1964). Subsequently Narahashi and Yanagita (1967) identified several distinct proteolytic activities including one which had aminopeptidase activity, and was activated by Ca2+ and Co2+. In contrast to other proteinase activities in the mixture, this aminopeptidase activity displayed considerable heat stablity (up to 80℃) and was unaffected by 9M urea. The activity, was, however, very sensitive to metal chelating agents.

While attempting to isolate the protein responsible for the trypsin activity in Pronase, Vosbeck ''et al.'' (1973) isolated two fractions with aminopeptidase activity. Although the two fractions differed slighty in their molecular weights (23K and 25K), they appeared to have the same enzymatic properties.

Interest in SGAP was renewed when aminopeptidases were recognized as useful tools in assays of metalloendopeptidase activity. The assay was based on a two stage reaction, with the endopeptidase cleaved an N-blocked peptide to release smaller peptide that was rapidly degraded by an aminopeptidase, generating a chromophore (Orlowski and Wilk, 1981; Mumford ''et al''., 1981). SGAP was considered an ideal tool for this purpose, given its stability, small size, and availability (Indig ''et al''., 1990).

== References ==


* Hiramatsu, A., & Ouchi, T. (1963). On the proteolytic enzymes from the commercial protease preparation of ''Streptomyces griseus'' (Pronase P). ''J. Biochem''. '''54''', 462-464.
* Indig, F.E., Benayahu, D., Fried, A., Wientroub, S., Blumberg, S. (1990). Neutral endopeptidase (EC 3.4.24.11) is highly expressed on osteoblastic cells and other marrow stromal cell types. ''Biochem. Biophys. Res. Commun''. '''172''', 620-626. [http://www.ncbi.nlm.nih.gov/pubmed/2241957?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 2241957]
* Mumford, R.A., Pierzchala, P.A., Strauss, A.W., Zimmerman, M. (1981). Purification of a membrane-bound metalloendopeptidase from porcine kidney that degrades peptide hormones. ''Proc. Natl Acad. Sci. USA'' '''78''', 6623-6627. [http://www.ncbi.nlm.nih.gov/pubmed/7031658?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7031658]
* Narahashi, Y., & Yanagita, M. (1967). Studies on proteolytic enzymes (Pronase) of S''treptomyces griseus'' K-1. I. Nature and properties of the proteolytic enzyme system. ''J. Biochem''. (Tokyo) '''62''', 633-641. [http://www.ncbi.nlm.nih.gov/pubmed/4968616?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4968616 ]
* Nomoto, M., & Narahashi, Y. (1959a). A proteolytic enzyme of ''Streptomyces griseus'': I. Purification of a protease of ''Streptomyces grisues''. ''J. Biochem.'' '''46''', 653-667.
* Nomoto, M., & Narahashi, Y. (1959b). A proteolytic enzyme of ''Streptomyces griseus'': III. Homogeneity of the purified enzyme preparation. ''J. Biochem.'' '''46''', 1481-1487.
* Nomoto, M., & Narahashi, Y. (1959c). A proteolytic enzyme of ''Streptomyces griseus'': IV. General properties of ''Streptomyces grisues'' protease. ''J. Biochem.'' '''46''', 1645-1651.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960a). A proteolytic enzyme of ''Streptomyces griseus'': V. Protective effect of calcium ion on the stability of protease. ''J. Biochem.'' '''48''', 453-463.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960b). A proteolytic enzyme of ''Streptomyces griseus'': VI. Hydrolysis of protein by ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 593-602.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960c). A proteolytic enzyme of ''Streptomyces griseus'': VII. Substrate specificity of ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 906-918.
* Nomoto, M., Narahashi, Y., Ouchi, T., & Hiramatsu, A. (1964). Abstract, 6th Inern. Congr. Biochem., N.Y., '''4''', 123.
* Orlowski, M., & Wilk, S. (1981). Purification and specificity of a membrane-bound metalloendopeptidase from bovine pituitaries. ''Biochemistry'' '''20''', 4942-4950. [http://www.ncbi.nlm.nih.gov/pubmed/7028098?ordinalpos=12&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7028098]
* Vosbeck, K. D., Chow, K. F., & Awad, W. M. Jr (1973). The proteolytic enzymes of the K-1 strain of ''Streptomyces griseus'' obtained from a commercial preparation (Pronase). Purification and characterization of the aminopeptidases. '' J. Biol. Chem''. '''248''', 6029-6034. [http://www.ncbi.nlm.nih.gov/pubmed/4199257?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4199257]

--[[User:Harry|Harry]] 15:20, 17 March 2008 (IST)

Nucleosomes

2008-05-15T10:14:25Z

Harry Greenblatt: /* Scenes for Projection in Biochemistry Classes */

For a general introduction beyond what is provided below, please see Wikipedia: [http://en.wikipedia.org/wiki/Nucleosomes Nucleosome]. For a list of nucleosome structures, see [[:Category:Nucleosome | Category: Nucleosome]].

==Scenes for Projection in Biochemistry Classes==

This section offers some large scenes of a nucleosome ([[1aoi]]), as determined in [http://www.mol.biol.ethz.ch/groups/richmond Tim Richmond's lab], suitable for projection in biochemistry classes.

<applet load="1aoi" size="450" color="white" frame="true" align="right" caption="1aoi, resolution 2.80Å" script="Nucleosomes/Rockets/8" />

<big>
1. '''<scene name='Nucleosomes/Composition/2'>Composition</scene>: DNA, Protein.'''

2. '''<scene name='Nucleosomes/Dna/3'>DNA</scene>'''

<big>Histone color key: '''H2a H2b H3 H4'''</big>

3. '''<scene name='Nucleosomes/Histones/4'>Histones</scene>'''

4. '''<scene name='Nucleosomes/Rockets/9'>Secondary Structure</scene>'''

<ul>
<li><scene name='Nucleosomes/Rockets/10'>H2a and H2b only</scene></li>
<li><scene name='Nucleosomes/Rockets/11'>H3 and H4 only</scene></li>
</ul>

</big>

 

==A Molecular Librarian==
This is an auspicious time for molecular biology. The wave of knowledge that began in 1944 with Avery's discovery of DNA as the genetic material, which lead naturally to the atomic model of DNA proposed by Watson and Crick, and continued through detailed experiments to determine the genetic code, is now cresting with the release of the first draft of the human genome. This molecular text, written through billions of years of evolution, will provide untold insights into the molecular processes that underlie every aspect of our lives.
[[Image:MotM Nucleosome 1aoi.gif |left]]

Each of our cells (or more correctly, nearly all of our cells) contain a copy of this genome, encoded in nine billion base pairs of DNA. This information is precious and must be carefully guarded. Inside our cells, a collection of repair enzymes correct chemical changes inflicted on the strands by environmental insults. But the delicate strands must also be protected from physical damage. This is the job of '''nucleosomes'''.

==A Paradox==
The job of the nucleosome is paradoxical, requiring it to perform two opposite functions simultaneously. On one hand, nucleosomes must be stable, forming tight, sheltering structures that compact the DNA and keep it from harm. On the other hand, nucleosomes must be labile enough to allow the information in the DNA to be used. Polymerases must be allowed access to the DNA, both to transcribe messenger RNA for building new proteins and to replicate the DNA when the cell divides. The method by which nucleosomes solve these opposed needs is not well understood, but may involve a partial unfolding of the DNA from around the nucleosome, one loop at a time, as the information in the DNA is read.

==Wagging Tails==
Apart from their function of safely packaging DNA, nucleosomes also modify the activity of the genes that they store. Each nucleosome is composed of eight "histone" proteins bundled tightly together at the center (shown here in blue), encircled by two loops of DNA (shown here in orange). The histone proteins, however, are not completely globular like most other proteins. They have long tails, which comprise nearly a quarter of their length. The tails extend outward from the compact nucleosome, reaching out to neighboring nucleosomes and binding them tightly together. The nucleus contains regulatory enzymes that chemically modify these tails to weaken their interactions. In this way, the cell makes particular genes more accessible to polymerases, allowing their particular information to be copied and used to build new proteins.

==Opposites Attract==
[[Image:MotM_Nucleosome_Nucleosome.gif | right]]
The histone proteins are perfectly designed for their jobs, so much so that histones are nearly identical in all non-bacterial organisms. Even slight modifications can be lethal. The surface of the histone octamer, shown on the left, is decorated with positively charged amino acids, shown with bright blue nitrogen atoms. These interact strongly with the negatively-charged phosphate groups on the DNA, shown at the right with bright yellow phosphorous and bright red oxygen atoms. This serves to glue the DNA strand to the protein core. This is no simple task. DNA is normally a long, straight molecule, but in nucleosomes the DNA must be forcably bent into these two tight circles.

==Exploring the Structure==
[[Image:1aoi_rasmol.gif | right]]
An intact nucleosome may be viewed in the PDB entry [[1aoi]]. The picture here shows the eight histone proteins as tubes that follow the protein chain, and shows the DNA as thinner tubes that follow the two strands as they circle around the protein octamer. The tails of the eight protein chains, seen extending outward from the center, are actually longer in reality. But, since they are so long and flexible, they are disordered in the crystal and cannot be seen. The one long chain at lower left gives a good indication of what the others might look like, if we were able to see the nucleosome inside a cell. Keep in mind that this structure only includes a short piece of DNA. In reality, these little nucleosomes are arrayed by the millions along long strands of DNA.

==Acknowledgements==
* Content adapted with permission from David S. Goodsell's [http://mgl.scripps.edu/people/goodsell/pdb/pdb7/pdb7_1.html Molecule of the Month on Nucleosomes].

[[Category:Nucleosome]]

Nucleosomes

2008-05-15T10:11:28Z

Harry Greenblatt: /* Scenes for Projection in Biochemistry Classes */

For a general introduction beyond what is provided below, please see Wikipedia: [http://en.wikipedia.org/wiki/Nucleosomes Nucleosome]. For a list of nucleosome structures, see [[:Category:Nucleosome | Category: Nucleosome]].

==Scenes for Projection in Biochemistry Classes==

This section offers some large scenes of a nucleosome ([[1aoi]]), as determined in [http://www.mol.biol.ethz.ch/groups/richmond Tim Richmond's lab], suitable for projection in biochemistry classes.

<applet load="1aoi" size="450" color="white" frame="true" align="right" caption="1aoi, resolution 2.80Å" script="Nucleosomes/Rockets/8" />

<big>
1. '''<scene name='Nucleosomes/Composition/2'>Composition</scene>: DNA, Protein.'''

2. '''<scene name='Nucleosomes/Dna/3'>DNA</scene>'''

<big>Histone color key: '''H2a H2b H3 H4'''</big>

3. '''<scene name='Nucleosomes/Histones/4'>Histones</scene>'''

4. '''<scene name='Nucleosomes/Rockets/9'>Secondary Structure</scene>'''

<ul>
<li><scene name='Nucleosomes/Rockets/10'>H2a and H2b only</scene></li>
<li><scene name='Nucleosomes/Rockets/6'>H3 and H4 only</scene></li>
</ul>

</big>

 

==A Molecular Librarian==
This is an auspicious time for molecular biology. The wave of knowledge that began in 1944 with Avery's discovery of DNA as the genetic material, which lead naturally to the atomic model of DNA proposed by Watson and Crick, and continued through detailed experiments to determine the genetic code, is now cresting with the release of the first draft of the human genome. This molecular text, written through billions of years of evolution, will provide untold insights into the molecular processes that underlie every aspect of our lives.
[[Image:MotM Nucleosome 1aoi.gif |left]]

Each of our cells (or more correctly, nearly all of our cells) contain a copy of this genome, encoded in nine billion base pairs of DNA. This information is precious and must be carefully guarded. Inside our cells, a collection of repair enzymes correct chemical changes inflicted on the strands by environmental insults. But the delicate strands must also be protected from physical damage. This is the job of '''nucleosomes'''.

==A Paradox==
The job of the nucleosome is paradoxical, requiring it to perform two opposite functions simultaneously. On one hand, nucleosomes must be stable, forming tight, sheltering structures that compact the DNA and keep it from harm. On the other hand, nucleosomes must be labile enough to allow the information in the DNA to be used. Polymerases must be allowed access to the DNA, both to transcribe messenger RNA for building new proteins and to replicate the DNA when the cell divides. The method by which nucleosomes solve these opposed needs is not well understood, but may involve a partial unfolding of the DNA from around the nucleosome, one loop at a time, as the information in the DNA is read.

==Wagging Tails==
Apart from their function of safely packaging DNA, nucleosomes also modify the activity of the genes that they store. Each nucleosome is composed of eight "histone" proteins bundled tightly together at the center (shown here in blue), encircled by two loops of DNA (shown here in orange). The histone proteins, however, are not completely globular like most other proteins. They have long tails, which comprise nearly a quarter of their length. The tails extend outward from the compact nucleosome, reaching out to neighboring nucleosomes and binding them tightly together. The nucleus contains regulatory enzymes that chemically modify these tails to weaken their interactions. In this way, the cell makes particular genes more accessible to polymerases, allowing their particular information to be copied and used to build new proteins.

==Opposites Attract==
[[Image:MotM_Nucleosome_Nucleosome.gif | right]]
The histone proteins are perfectly designed for their jobs, so much so that histones are nearly identical in all non-bacterial organisms. Even slight modifications can be lethal. The surface of the histone octamer, shown on the left, is decorated with positively charged amino acids, shown with bright blue nitrogen atoms. These interact strongly with the negatively-charged phosphate groups on the DNA, shown at the right with bright yellow phosphorous and bright red oxygen atoms. This serves to glue the DNA strand to the protein core. This is no simple task. DNA is normally a long, straight molecule, but in nucleosomes the DNA must be forcably bent into these two tight circles.

==Exploring the Structure==
[[Image:1aoi_rasmol.gif | right]]
An intact nucleosome may be viewed in the PDB entry [[1aoi]]. The picture here shows the eight histone proteins as tubes that follow the protein chain, and shows the DNA as thinner tubes that follow the two strands as they circle around the protein octamer. The tails of the eight protein chains, seen extending outward from the center, are actually longer in reality. But, since they are so long and flexible, they are disordered in the crystal and cannot be seen. The one long chain at lower left gives a good indication of what the others might look like, if we were able to see the nucleosome inside a cell. Keep in mind that this structure only includes a short piece of DNA. In reality, these little nucleosomes are arrayed by the millions along long strands of DNA.

==Acknowledgements==
* Content adapted with permission from David S. Goodsell's [http://mgl.scripps.edu/people/goodsell/pdb/pdb7/pdb7_1.html Molecule of the Month on Nucleosomes].

[[Category:Nucleosome]]

Nucleosomes

2008-05-15T10:06:56Z

Harry Greenblatt: /* Scenes for Projection in Biochemistry Classes */

For a general introduction beyond what is provided below, please see Wikipedia: [http://en.wikipedia.org/wiki/Nucleosomes Nucleosome]. For a list of nucleosome structures, see [[:Category:Nucleosome | Category: Nucleosome]].

==Scenes for Projection in Biochemistry Classes==

This section offers some large scenes of a nucleosome ([[1aoi]]), as determined in [http://www.mol.biol.ethz.ch/groups/richmond Tim Richmond's lab], suitable for projection in biochemistry classes.

<applet load="1aoi" size="450" color="white" frame="true" align="right" caption="1aoi, resolution 2.80Å" script="Nucleosomes/Rockets/8" />

<big>
1. '''<scene name='Nucleosomes/Composition/2'>Composition</scene>: DNA, Protein.'''

2. '''<scene name='Nucleosomes/Dna/3'>DNA</scene>'''

<big>Histone color key: '''H2a H2b H3 H4'''</big>

3. '''<scene name='Nucleosomes/Histones/4'>Histones</scene>'''

4. '''<scene name='Nucleosomes/Rockets/9'>Secondary Structure</scene>'''

<ul>
<li><scene name='Nucleosomes/Rockets/5'>H2a and H2b only</scene></li>
<li><scene name='Nucleosomes/Rockets/6'>H3 and H4 only</scene></li>
</ul>

</big>

 

==A Molecular Librarian==
This is an auspicious time for molecular biology. The wave of knowledge that began in 1944 with Avery's discovery of DNA as the genetic material, which lead naturally to the atomic model of DNA proposed by Watson and Crick, and continued through detailed experiments to determine the genetic code, is now cresting with the release of the first draft of the human genome. This molecular text, written through billions of years of evolution, will provide untold insights into the molecular processes that underlie every aspect of our lives.
[[Image:MotM Nucleosome 1aoi.gif |left]]

Each of our cells (or more correctly, nearly all of our cells) contain a copy of this genome, encoded in nine billion base pairs of DNA. This information is precious and must be carefully guarded. Inside our cells, a collection of repair enzymes correct chemical changes inflicted on the strands by environmental insults. But the delicate strands must also be protected from physical damage. This is the job of '''nucleosomes'''.

==A Paradox==
The job of the nucleosome is paradoxical, requiring it to perform two opposite functions simultaneously. On one hand, nucleosomes must be stable, forming tight, sheltering structures that compact the DNA and keep it from harm. On the other hand, nucleosomes must be labile enough to allow the information in the DNA to be used. Polymerases must be allowed access to the DNA, both to transcribe messenger RNA for building new proteins and to replicate the DNA when the cell divides. The method by which nucleosomes solve these opposed needs is not well understood, but may involve a partial unfolding of the DNA from around the nucleosome, one loop at a time, as the information in the DNA is read.

==Wagging Tails==
Apart from their function of safely packaging DNA, nucleosomes also modify the activity of the genes that they store. Each nucleosome is composed of eight "histone" proteins bundled tightly together at the center (shown here in blue), encircled by two loops of DNA (shown here in orange). The histone proteins, however, are not completely globular like most other proteins. They have long tails, which comprise nearly a quarter of their length. The tails extend outward from the compact nucleosome, reaching out to neighboring nucleosomes and binding them tightly together. The nucleus contains regulatory enzymes that chemically modify these tails to weaken their interactions. In this way, the cell makes particular genes more accessible to polymerases, allowing their particular information to be copied and used to build new proteins.

==Opposites Attract==
[[Image:MotM_Nucleosome_Nucleosome.gif | right]]
The histone proteins are perfectly designed for their jobs, so much so that histones are nearly identical in all non-bacterial organisms. Even slight modifications can be lethal. The surface of the histone octamer, shown on the left, is decorated with positively charged amino acids, shown with bright blue nitrogen atoms. These interact strongly with the negatively-charged phosphate groups on the DNA, shown at the right with bright yellow phosphorous and bright red oxygen atoms. This serves to glue the DNA strand to the protein core. This is no simple task. DNA is normally a long, straight molecule, but in nucleosomes the DNA must be forcably bent into these two tight circles.

==Exploring the Structure==
[[Image:1aoi_rasmol.gif | right]]
An intact nucleosome may be viewed in the PDB entry [[1aoi]]. The picture here shows the eight histone proteins as tubes that follow the protein chain, and shows the DNA as thinner tubes that follow the two strands as they circle around the protein octamer. The tails of the eight protein chains, seen extending outward from the center, are actually longer in reality. But, since they are so long and flexible, they are disordered in the crystal and cannot be seen. The one long chain at lower left gives a good indication of what the others might look like, if we were able to see the nucleosome inside a cell. Keep in mind that this structure only includes a short piece of DNA. In reality, these little nucleosomes are arrayed by the millions along long strands of DNA.

==Acknowledgements==
* Content adapted with permission from David S. Goodsell's [http://mgl.scripps.edu/people/goodsell/pdb/pdb7/pdb7_1.html Molecule of the Month on Nucleosomes].

[[Category:Nucleosome]]

Nucleosomes

2008-05-15T09:59:27Z

Harry Greenblatt: /* Scenes for Projection in Biochemistry Classes */

For a general introduction beyond what is provided below, please see Wikipedia: [http://en.wikipedia.org/wiki/Nucleosomes Nucleosome]. For a list of nucleosome structures, see [[:Category:Nucleosome | Category: Nucleosome]].

==Scenes for Projection in Biochemistry Classes==

This section offers some large scenes of a nucleosome ([[1aoi]]), as determined in [http://www.mol.biol.ethz.ch/groups/richmond Tim Richmond's lab], suitable for projection in biochemistry classes.

<applet load="1aoi" size="450" color="white" frame="true" align="right" caption="1aoi, resolution 2.80Å" script="Nucleosomes/Rockets/8" />

<big>
1. '''<scene name='Nucleosomes/Composition/2'>Composition</scene>: DNA, Protein.'''

2. '''<scene name='Nucleosomes/Dna/3'>DNA</scene>'''

<big>Histone color key: '''H2a H2b H3 H4'''</big>

3. '''<scene name='Nucleosomes/Histones/4'>Histones</scene>'''

4. '''<scene name='Nucleosomes/Rockets/4'>Secondary Structure</scene>'''

<ul>
<li><scene name='Nucleosomes/Rockets/5'>H2a and H2b only</scene></li>
<li><scene name='Nucleosomes/Rockets/6'>H3 and H4 only</scene></li>
</ul>

</big>

 

==A Molecular Librarian==
This is an auspicious time for molecular biology. The wave of knowledge that began in 1944 with Avery's discovery of DNA as the genetic material, which lead naturally to the atomic model of DNA proposed by Watson and Crick, and continued through detailed experiments to determine the genetic code, is now cresting with the release of the first draft of the human genome. This molecular text, written through billions of years of evolution, will provide untold insights into the molecular processes that underlie every aspect of our lives.
[[Image:MotM Nucleosome 1aoi.gif |left]]

Each of our cells (or more correctly, nearly all of our cells) contain a copy of this genome, encoded in nine billion base pairs of DNA. This information is precious and must be carefully guarded. Inside our cells, a collection of repair enzymes correct chemical changes inflicted on the strands by environmental insults. But the delicate strands must also be protected from physical damage. This is the job of '''nucleosomes'''.

==A Paradox==
The job of the nucleosome is paradoxical, requiring it to perform two opposite functions simultaneously. On one hand, nucleosomes must be stable, forming tight, sheltering structures that compact the DNA and keep it from harm. On the other hand, nucleosomes must be labile enough to allow the information in the DNA to be used. Polymerases must be allowed access to the DNA, both to transcribe messenger RNA for building new proteins and to replicate the DNA when the cell divides. The method by which nucleosomes solve these opposed needs is not well understood, but may involve a partial unfolding of the DNA from around the nucleosome, one loop at a time, as the information in the DNA is read.

==Wagging Tails==
Apart from their function of safely packaging DNA, nucleosomes also modify the activity of the genes that they store. Each nucleosome is composed of eight "histone" proteins bundled tightly together at the center (shown here in blue), encircled by two loops of DNA (shown here in orange). The histone proteins, however, are not completely globular like most other proteins. They have long tails, which comprise nearly a quarter of their length. The tails extend outward from the compact nucleosome, reaching out to neighboring nucleosomes and binding them tightly together. The nucleus contains regulatory enzymes that chemically modify these tails to weaken their interactions. In this way, the cell makes particular genes more accessible to polymerases, allowing their particular information to be copied and used to build new proteins.

==Opposites Attract==
[[Image:MotM_Nucleosome_Nucleosome.gif | right]]
The histone proteins are perfectly designed for their jobs, so much so that histones are nearly identical in all non-bacterial organisms. Even slight modifications can be lethal. The surface of the histone octamer, shown on the left, is decorated with positively charged amino acids, shown with bright blue nitrogen atoms. These interact strongly with the negatively-charged phosphate groups on the DNA, shown at the right with bright yellow phosphorous and bright red oxygen atoms. This serves to glue the DNA strand to the protein core. This is no simple task. DNA is normally a long, straight molecule, but in nucleosomes the DNA must be forcably bent into these two tight circles.

==Exploring the Structure==
[[Image:1aoi_rasmol.gif | right]]
An intact nucleosome may be viewed in the PDB entry [[1aoi]]. The picture here shows the eight histone proteins as tubes that follow the protein chain, and shows the DNA as thinner tubes that follow the two strands as they circle around the protein octamer. The tails of the eight protein chains, seen extending outward from the center, are actually longer in reality. But, since they are so long and flexible, they are disordered in the crystal and cannot be seen. The one long chain at lower left gives a good indication of what the others might look like, if we were able to see the nucleosome inside a cell. Keep in mind that this structure only includes a short piece of DNA. In reality, these little nucleosomes are arrayed by the millions along long strands of DNA.

==Acknowledgements==
* Content adapted with permission from David S. Goodsell's [http://mgl.scripps.edu/people/goodsell/pdb/pdb7/pdb7_1.html Molecule of the Month on Nucleosomes].

[[Category:Nucleosome]]

Nucleosomes

2008-05-15T09:49:34Z

Harry Greenblatt: /* Scenes for Projection in Biochemistry Classes */

For a general introduction beyond what is provided below, please see Wikipedia: [http://en.wikipedia.org/wiki/Nucleosomes Nucleosome]. For a list of nucleosome structures, see [[:Category:Nucleosome | Category: Nucleosome]].

==Scenes for Projection in Biochemistry Classes==

This section offers some large scenes of a nucleosome ([[1aoi]]), as determined in [http://www.mol.biol.ethz.ch/groups/richmond Tim Richmond's lab], suitable for projection in biochemistry classes.

<applet load="1aoi" size="450" color="white" frame="true" align="right" caption="1aoi, resolution 2.80Å" script="Nucleosomes/Rockets/8" />

<big>
1. '''<scene name='Nucleosomes/Composition/2'>Composition</scene>: DNA, Protein.'''

2. '''<scene name='Nucleosomes/Dna/3'>DNA</scene>'''

<big>Histone color key: '''H2a H2b H3 H4'''</big>

3. '''<scene name='Nucleosomes/Histones/3'>Histones</scene>'''

4. '''<scene name='Nucleosomes/Rockets/4'>Secondary Structure</scene>'''

<ul>
<li><scene name='Nucleosomes/Rockets/5'>H2a and H2b only</scene></li>
<li><scene name='Nucleosomes/Rockets/6'>H3 and H4 only</scene></li>
</ul>

</big>

 

==A Molecular Librarian==
This is an auspicious time for molecular biology. The wave of knowledge that began in 1944 with Avery's discovery of DNA as the genetic material, which lead naturally to the atomic model of DNA proposed by Watson and Crick, and continued through detailed experiments to determine the genetic code, is now cresting with the release of the first draft of the human genome. This molecular text, written through billions of years of evolution, will provide untold insights into the molecular processes that underlie every aspect of our lives.
[[Image:MotM Nucleosome 1aoi.gif |left]]

Each of our cells (or more correctly, nearly all of our cells) contain a copy of this genome, encoded in nine billion base pairs of DNA. This information is precious and must be carefully guarded. Inside our cells, a collection of repair enzymes correct chemical changes inflicted on the strands by environmental insults. But the delicate strands must also be protected from physical damage. This is the job of '''nucleosomes'''.

==A Paradox==
The job of the nucleosome is paradoxical, requiring it to perform two opposite functions simultaneously. On one hand, nucleosomes must be stable, forming tight, sheltering structures that compact the DNA and keep it from harm. On the other hand, nucleosomes must be labile enough to allow the information in the DNA to be used. Polymerases must be allowed access to the DNA, both to transcribe messenger RNA for building new proteins and to replicate the DNA when the cell divides. The method by which nucleosomes solve these opposed needs is not well understood, but may involve a partial unfolding of the DNA from around the nucleosome, one loop at a time, as the information in the DNA is read.

==Wagging Tails==
Apart from their function of safely packaging DNA, nucleosomes also modify the activity of the genes that they store. Each nucleosome is composed of eight "histone" proteins bundled tightly together at the center (shown here in blue), encircled by two loops of DNA (shown here in orange). The histone proteins, however, are not completely globular like most other proteins. They have long tails, which comprise nearly a quarter of their length. The tails extend outward from the compact nucleosome, reaching out to neighboring nucleosomes and binding them tightly together. The nucleus contains regulatory enzymes that chemically modify these tails to weaken their interactions. In this way, the cell makes particular genes more accessible to polymerases, allowing their particular information to be copied and used to build new proteins.

==Opposites Attract==
[[Image:MotM_Nucleosome_Nucleosome.gif | right]]
The histone proteins are perfectly designed for their jobs, so much so that histones are nearly identical in all non-bacterial organisms. Even slight modifications can be lethal. The surface of the histone octamer, shown on the left, is decorated with positively charged amino acids, shown with bright blue nitrogen atoms. These interact strongly with the negatively-charged phosphate groups on the DNA, shown at the right with bright yellow phosphorous and bright red oxygen atoms. This serves to glue the DNA strand to the protein core. This is no simple task. DNA is normally a long, straight molecule, but in nucleosomes the DNA must be forcably bent into these two tight circles.

==Exploring the Structure==
[[Image:1aoi_rasmol.gif | right]]
An intact nucleosome may be viewed in the PDB entry [[1aoi]]. The picture here shows the eight histone proteins as tubes that follow the protein chain, and shows the DNA as thinner tubes that follow the two strands as they circle around the protein octamer. The tails of the eight protein chains, seen extending outward from the center, are actually longer in reality. But, since they are so long and flexible, they are disordered in the crystal and cannot be seen. The one long chain at lower left gives a good indication of what the others might look like, if we were able to see the nucleosome inside a cell. Keep in mind that this structure only includes a short piece of DNA. In reality, these little nucleosomes are arrayed by the millions along long strands of DNA.

==Acknowledgements==
* Content adapted with permission from David S. Goodsell's [http://mgl.scripps.edu/people/goodsell/pdb/pdb7/pdb7_1.html Molecule of the Month on Nucleosomes].

[[Category:Nucleosome]]

User:Harry Greenblatt

2008-04-07T11:28:52Z

Harry Greenblatt:

<applet load="4sgb" size="300" color="white" frame="true" align="right" caption="4sgb, Polypeptide Chymotrypsin Inhibitor I, bound to SGPB" scene="User:Harry/4sgb/1"/>

Dr. Harry M. Greenblatt 
Associate Staff Scientist 
Department of Structural Biology 
Faculty of Chemistry 
Weizmann Institute of Science 
Rehovot, Israel 
76100

Office: 972-(0)8-934-3625

Harry#dot#Greenblatt#atsymbol#weizmann#dot#ac#dot#il

User:Harry Greenblatt

2008-03-19T09:07:07Z

Harry Greenblatt: PCI-1 from Russet Burbank Potato Tubers bound to Streptomyces griseus Proteinase B

<applet load="4sgb" size="300" color="white" frame="true" align="right" caption="4sgb, Polypeptide Chymotrypsin Inhibitor I, bound to SGPB" scene="User:Harry/4sgb/1"/>

Dr. Harry M. Greenblatt 
Assistant Staff Scientist 
Department of Structural Biology 
Faculty of Chemistry 
Weizmann Institute of Science 
Rehovot, Israel 
76100

Office: 972-(0)8-934-3625

Harry#dot#Greenblatt#atsymbol#weizmann#dot#ac#dot#il

User:Harry Greenblatt

2008-03-19T08:53:37Z

Harry Greenblatt:

<applet load="4sgb" size="300" color="white" frame="true" align="right" caption="4sgb, Polypeptide Chymotrypsin Inhibitor I, bound to SGPB"/>

Dr. Harry M. Greenblatt 
Assistant Staff Scientist 
Department of Structural Biology 
Faculty of Chemistry 
Weizmann Institute of Science 
Rehovot, Israel 
76100

Office: 972-(0)8-934-3625

Harry#dot#Greenblatt#atsymbol#weizmann#dot#ac#dot#il

User:Harry Greenblatt

2008-03-19T08:52:30Z

Harry Greenblatt:

<applet load="4sgb" size="300" color="white" frame="true" align="right" caption="4sgb, Potato Chymotrypsin Inhibitor I, bound to SGPB"/>

Dr. Harry M. Greenblatt 
Assistant Staff Scientist 
Department of Structural Biology 
Faculty of Chemistry 
Weizmann Institute of Science 
Rehovot, Israel 
76100

Office: 972-(0)8-934-3625

Harry#dot#Greenblatt#atsymbol#weizmann#dot#ac#dot#il

User:Harry Greenblatt

2008-03-19T08:12:50Z

Harry Greenblatt: New page: <applet load="166d" size="300" color="white" frame="true" align="right" /> Dr. Harry M. Greenblatt Assistant Staff Scientist Department of Structural Biology Faculty of C...

<applet load="166d" size="300" color="white" frame="true" align="right" />

Dr. Harry M. Greenblatt 
Assistant Staff Scientist 
Department of Structural Biology 
Faculty of Chemistry 
Weizmann Institute of Science 
Rehovot, Israel 
76100

Office: 972-(0)8-934-3625

Harry#dot#Greenblatt#atsymbol#weizmann#dot#ac#dot#il

File:Soman reaction.png

2008-03-18T11:32:37Z

Harry Greenblatt: Reaction between Ser200OG and Soman, assuming an in-line attack by the OG. "Aging" is caused by spontaneous dealkylation of the O-pinacolyl group.

Reaction between Ser200OG and Soman, assuming an in-line attack by the OG. "Aging" is caused by spontaneous dealkylation of the O-pinacolyl group.

Acetylcholinesterase

2008-03-17T13:54:10Z

Harry Greenblatt: /* Selected 3D Structures of AChE */

[[Image:small_wh_ray0001.gif|left|150px]] 
<applet load="1ea5_rot.pdb" size="300" color="white" frame="true" spin="on" caption="AChE" align="right" script="Acetylcholinesterase/New_down_gorge/1" />
'''3D structure of acetylcholinesterase''' 
== Key Enzyme in the Nervous System ==

'''Acetylcholinesterase''' (AChE) is key enzyme in the nervous system of animals. By rapid hydrolysis of the neurotransmitter, '''acetylcholine''' (ACh), AChE terminates neurotransmission at cholinergic synapses. It is a very fast enzyme, especially for a serine hydrolase, functioning at a rate approaching that of a diffusion-controlled reaction. AChE inhibitors are among the key drugs approved by the FDA for management of Alzheimer's disease (AD). The powerful toxicity of organophosphorus (OP) poisons is attributed primarily to their potent AChE inhibitors.
[[Image:Synapse_Schematic.jpg|thumb|Cholinergic Synapse|300px|left]]

The 3D structure of ''Torpedo californica'' AChE (''Tc''AChE) ([http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1678899 Sussman et al. & Silman (1991)]) opened up new horizons in research on an enzyme that had already been the subject of intensive investigation. The unanticipated structure of this extremely rapid enzyme, in which the active site was found to be buried at the bottom of a
<scene name='Acetylcholinesterase/New_down_gorge/2'>deep and narrow gorge</scene>,
lined by aromatic residues, led to a revision of the views then held concerning substrate traffic, recognition,
and hydrolysis ([http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10545346 Botti et al. Sussman & Silman (1999)]).

Alzheimer’s disease (AD) is a debilitating brain disease that occurs in around 10% of the elderly and, as yet, there is no known cure. At present, the most widely used treatments consist are medications that attempt to increase the brain’s levels of ACh, whose levels decrease with onset of disease. These drugs work by interfering with AChE. Thus drugs that are mild inhibitors of AChE, like Tacrine, E2020 (Aricept) and the Traditonal Chinese Medicine (TCM) Huperzine appear to retard symptoms of AD.

<applet load='1ea5_rot.pdb' size='300' color='white' frame='true' spin='on' caption='AChE' align='right' script='Acetylcholinesterase/New_down_gorge/5'
'''3D structure of acetylcholinesterase''' 
The active site gorge has <scene name='Acetylcholinesterase/New_down_gorge/6'>two binding sites</scene>, a catalytic site (consisting of the catalytic triad together with Trp84 & Phe330) and a peripheral site (including Trp 279 & Tyr 121), which helps prebind the substrate and direct it toward the active site. The 3D structure showed not only that the active site was buried deep in the enzyme, but surprisingly, there were no negatively charged residues along this gorge, as was expected to help attract the positively charged ACh substrate, rather, instead, a series of aromatic residues that are highly conserved in all AChE sequences. See: [[AChE inhibitors and substrates]]

==Selected 3D Structures of AChE ==
* [[2ace]] This is the original solved structure for '''''T. californica'''''
* [[1ea5]] This is the highest resolution X-ray structure of AChE determined till now.
* [[1eve]] The E2020 (Aricept) complex.
* [[1ax9]] Edrophonium complex.
* [[1vot]] Complex with huperzine, a traditional Chinese folk medicine.
* [[1fss]] Complex with the snake venom toxin, Fasciculin-II.
* [[1vzj]] Model complex of the cholinesterase tetramer.
* [[1som]] Complex with nerve agent soman (GD).

More structures can be obtained by searching for
[http://proteopedia.org/wiki/index.php/Special:Search?search=AChE&fulltext=AChE AChE]

[[Category: catalytic triad]]
[[Category: cholinesterase]]
[[Category: cholinesterases]]
[[Category: acetylcholine]]
[[Category: cation-pi]]
[[Category: Alzheimers]]
[[Category: nerve gasses]]

Acetylcholinesterase

2008-03-17T13:52:58Z

Harry Greenblatt: add 1som, fix up list of selected structures to make case consistent.

[[Image:small_wh_ray0001.gif|left|150px]] 
<applet load="1ea5_rot.pdb" size="300" color="white" frame="true" spin="on" caption="AChE" align="right" script="Acetylcholinesterase/New_down_gorge/1" />
'''3D structure of acetylcholinesterase''' 
== Key Enzyme in the Nervous System ==

'''Acetylcholinesterase''' (AChE) is key enzyme in the nervous system of animals. By rapid hydrolysis of the neurotransmitter, '''acetylcholine''' (ACh), AChE terminates neurotransmission at cholinergic synapses. It is a very fast enzyme, especially for a serine hydrolase, functioning at a rate approaching that of a diffusion-controlled reaction. AChE inhibitors are among the key drugs approved by the FDA for management of Alzheimer's disease (AD). The powerful toxicity of organophosphorus (OP) poisons is attributed primarily to their potent AChE inhibitors.
[[Image:Synapse_Schematic.jpg|thumb|Cholinergic Synapse|300px|left]]

The 3D structure of ''Torpedo californica'' AChE (''Tc''AChE) ([http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1678899 Sussman et al. & Silman (1991)]) opened up new horizons in research on an enzyme that had already been the subject of intensive investigation. The unanticipated structure of this extremely rapid enzyme, in which the active site was found to be buried at the bottom of a
<scene name='Acetylcholinesterase/New_down_gorge/2'>deep and narrow gorge</scene>,
lined by aromatic residues, led to a revision of the views then held concerning substrate traffic, recognition,
and hydrolysis ([http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10545346 Botti et al. Sussman & Silman (1999)]).

Alzheimer’s disease (AD) is a debilitating brain disease that occurs in around 10% of the elderly and, as yet, there is no known cure. At present, the most widely used treatments consist are medications that attempt to increase the brain’s levels of ACh, whose levels decrease with onset of disease. These drugs work by interfering with AChE. Thus drugs that are mild inhibitors of AChE, like Tacrine, E2020 (Aricept) and the Traditonal Chinese Medicine (TCM) Huperzine appear to retard symptoms of AD.

<applet load='1ea5_rot.pdb' size='300' color='white' frame='true' spin='on' caption='AChE' align='right' script='Acetylcholinesterase/New_down_gorge/5'
'''3D structure of acetylcholinesterase''' 
The active site gorge has <scene name='Acetylcholinesterase/New_down_gorge/6'>two binding sites</scene>, a catalytic site (consisting of the catalytic triad together with Trp84 & Phe330) and a peripheral site (including Trp 279 & Tyr 121), which helps prebind the substrate and direct it toward the active site. The 3D structure showed not only that the active site was buried deep in the enzyme, but surprisingly, there were no negatively charged residues along this gorge, as was expected to help attract the positively charged ACh substrate, rather, instead, a series of aromatic residues that are highly conserved in all AChE sequences. See: [[AChE inhibitors and substrates]]

==Selected 3D Structures of AChE ==
* [[2ace]] This is the original solved structure for '''''T. californica'''''
* [[1ea5]] This is the highest resolution X-ray structure of AChE determined till now.
* [[1eve]] The E2020 (Aricept) complex.
* [[1ax9]] Edrophonium complex.
* [[1vot]] Complex with huperzine, a traditional Chinese folk medicine.
* [[1fss]] Complex with the snake venom toxin, Fasciculin-II.
* [[1vzj]] Model complex of the Cholinesterase tetramer.
* [[1som]] Complex with nerve agent soman (GD).

More structures can be obtained by searching for
[http://proteopedia.org/wiki/index.php/Special:Search?search=AChE&fulltext=AChE AChE]

[[Category: catalytic triad]]
[[Category: cholinesterase]]
[[Category: cholinesterases]]
[[Category: acetylcholine]]
[[Category: cation-pi]]
[[Category: Alzheimers]]
[[Category: nerve gasses]]

Streptomyces griseus Aminopeptidase (SGAP)

2008-03-17T13:33:07Z

Harry Greenblatt:

[[Image:1xjo_cartoon.png | 330px | thumb | S griseus aminopeptidase, showing overall fold. Zinc ions are dark grey, calcium ion is white.]]
'''Title''' : Structure of ''S. griseus'' aminopeptidase

== ''Streptomyces griseus'' Aminopeptidase (SGAP) ==

==Biological function==
''S. griseus'' Aminopeptidase (SGAP; E.C. 3.4.11.-) cleaves the N-terminal amino acid from a peptide or protein, and is specific for larger hydrophobic acids, especially leucine. No cleavage occurs if the next residue is proline.

[[Image:aminopeptidase_rxn2.png | left | thumb| 800px | Reaction catalyzed by SGAP; scissile bond is shown in red.]]
 

== Biological Context ==

SGAP is one of the many proteinases present in the extracellular fluid of cultures of Streptomyces griseus, and can be isolated from [http://en.wikipedia.org/wiki/Pronase Pronase], the commercial preparation of the extracellular fluid from this organism. SGAP is a monomeric, 30KDa, heat stable enzyme requiring two Zn2+ ions for activity, and is activated by Ca2+.

== Structures Available ==

* [[1xjo]] - native SGAP to 1.75Å
* [[1cp7]] - native SGAP to 1.58Å
* [[1qq9]] - SGAP complexed with L-methionine to 1.53Å
* [[1f2o]] - complex with L-leucine to 1.70Å
* [[1f2p]] - complex with L-phenylalanine to 1.80Å
* [[1tf8]] - complex with L-tryptophane to 1.30Å
* [[1tf9]] - complex with ''p''-iodo-L-phenylalanine to 1.30Å
* [[1tkf]] - with D-tryptophan to 1.20Å
* [[1tkh]] - with D-phenylalanine to 1.25Å
* [[1tkj]] - with D-methionine to 1.15Å
* [[1xbu]] - with ''p''-iodo-D-phenylalanine to 1.20Å

== Historical Context ==

The proteolytic activity contained in the extracellular fluid of cultures of ''Streptomyces griseus'' was first identified by Nomoto and Narahashi (1959a), who obtained a highly purified preparation of this activity from the K-1 strain of this bactreria. A large scale version of their procedure was used to prepare commercial quantities of this preparation (Pronase). Various physical criteria showed that Pronase was homogeneous (Nomoto and Narahashi, 1959b), yet displayed both exopeptidase and endopeptidase activity, with a wide range of side chain specificities (Nomoto and Narahashi, 1959b, 1959c; Nomoto ''et al''., 1960a, 1960b; 1960c). The supposed homogeneity of Pronase was controversial, with other investigators using various chromatographic methods to isolate more fractions with proteolytic activity (Hiramatsu and Ouchi, 1963,; Nomoto et al., 1964). Subsequently Narahashi and Yanagita (1967) identified several distinct proteolytic activities including one which had aminopeptidase activity, and was activated by Ca2+ and Co2+. In contrast to other proteinase activities in the mixture, this aminopeptidase activity displayed considerable heat stablity (up to 80℃) and was unaffected by 9M urea. The activity, was, however, very sensitive to metal chelating agents.

While attempting to isolate the protein responsible for the trypsin activity in Pronase, Vosbeck ''et al.'' (1973) isolated two fractions with aminopeptidase activity. Although the two fractions differed slighty in their molecular weights (23K and 25K), they appeared to have the same enzymatic properties.

Interest in SGAP was renewed when aminopeptidases were recognized as useful tools in assays of metalloendopeptidase activity. The assay was based on a two stage reaction, with the endopeptidase cleaved an N-blocked peptide to release smaller peptide that was rapidly degraded by an aminopeptidase, generating a chromophore (Orlowski and Wilk, 1981; Mumford ''et al''., 1981). SGAP was considered an ideal tool for this purpose, given its stability, small size, and availability (Indig ''et al''., 1990).

== References ==


* Hiramatsu, A., & Ouchi, T. (1963). On the proteolytic enzymes from the commercial protease preparation of ''Streptomyces griseus'' (Pronase P). ''J. Biochem''. '''54''', 462-464.
* Indig, F.E., Benayahu, D., Fried, A., Wientroub, S., Blumberg, S. (1990). Neutral endopeptidase (EC 3.4.24.11) is highly expressed on osteoblastic cells and other marrow stromal cell types. ''Biochem. Biophys. Res. Commun''. '''172''', 620-626. [http://www.ncbi.nlm.nih.gov/pubmed/2241957?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 2241957]
* Mumford, R.A., Pierzchala, P.A., Strauss, A.W., Zimmerman, M. (1981). Purification of a membrane-bound metalloendopeptidase from porcine kidney that degrades peptide hormones. ''Proc. Natl Acad. Sci. USA'' '''78''', 6623-6627. [http://www.ncbi.nlm.nih.gov/pubmed/7031658?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7031658]
* Narahashi, Y., & Yanagita, M. (1967). Studies on proteolytic enzymes (Pronase) of S''treptomyces griseus'' K-1. I. Nature and properties of the proteolytic enzyme system. ''J. Biochem''. (Tokyo) '''62''', 633-641. [http://www.ncbi.nlm.nih.gov/pubmed/4968616?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4968616 ]
* Nomoto, M., & Narahashi, Y. (1959a). A proteolytic enzyme of ''Streptomyces griseus'': I. Purification of a protease of ''Streptomyces grisues''. ''J. Biochem.'' '''46''', 653-667.
* Nomoto, M., & Narahashi, Y. (1959b). A proteolytic enzyme of ''Streptomyces griseus'': III. Homogeneity of the purified enzyme preparation. ''J. Biochem.'' '''46''', 1481-1487.
* Nomoto, M., & Narahashi, Y. (1959c). A proteolytic enzyme of ''Streptomyces griseus'': IV. General properties of ''Streptomyces grisues'' protease. ''J. Biochem.'' '''46''', 1645-1651.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960a). A proteolytic enzyme of ''Streptomyces griseus'': V. Protective effect of calcium ion on the stability of protease. ''J. Biochem.'' '''48''', 453-463.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960b). A proteolytic enzyme of ''Streptomyces griseus'': VI. Hydrolysis of protein by ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 593-602.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960c). A proteolytic enzyme of ''Streptomyces griseus'': VII. Substrate specificity of ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 906-918.
* Nomoto, M., Narahashi, Y., Ouchi, T., & Hiramatsu, A. (1964). Abstract, 6th Inern. Congr. Biochem., N.Y., '''4''', 123.
* Orlowski, M., & Wilk, S. (1981). Purification and specificity of a membrane-bound metalloendopeptidase from bovine pituitaries. ''Biochemistry'' '''20''', 4942-4950. [http://www.ncbi.nlm.nih.gov/pubmed/7028098?ordinalpos=12&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7028098]
* Vosbeck, K. D., Chow, K. F., & Awad, W. M. Jr (1973). The proteolytic enzymes of the K-1 strain of ''Streptomyces griseus'' obtained from a commercial preparation (Pronase). Purification and characterization of the aminopeptidases. '' J. Biol. Chem''. '''248''', 6029-6034. [http://www.ncbi.nlm.nih.gov/pubmed/4199257?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4199257]

--[[User:Harry|Harry]] 15:20, 17 March 2008 (IST)

Streptomyces griseus Aminopeptidase (SGAP)

2008-03-17T13:20:58Z

Harry Greenblatt:

== ''Streptomyces griseus'' Aminopeptidase (SGAP) ==

==Biological function==
''S. griseus'' Aminopeptidase (SGAP; E.C. 3.4.11.-) cleaves the N-terminal amino acid from a peptide or protein, and is specific for larger hydrophobic acids, especially leucine. No cleavage occurs if the next residue is proline.

[[Image:aminopeptidase_rxn2.png | left | thumb| 800px | Reaction catalyzed by SGAP; scissile bond is shown in red.]]
 

== Biological Context ==

SGAP is one of the many proteinases present in the extracellular fluid of cultures of Streptomyces griseus, and can be isolated from [http://en.wikipedia.org/wiki/Pronase Pronase], the commercial preparation of the extracellular fluid from this organism. SGAP is a monomeric, 30KDa, heat stable enzyme requiring two Zn2+ ions for activity, and is activated by Ca2+.

== Structures Available ==

* [[1xjo]] - native SGAP to 1.75Å
* [[1cp7]] - native SGAP to 1.58Å
* [[1qq9]] - SGAP complexed with L-methionine to 1.53Å
* [[1f2o]] - complex with L-leucine to 1.70Å
* [[1f2p]] - complex with L-phenylalanine to 1.80Å
* [[1tf8]] - complex with L-tryptophane to 1.30Å
* [[1tf9]] - complex with ''p''-iodo-L-phenylalanine to 1.30Å
* [[1tkf]] - with D-tryptophan to 1.20Å
* [[1tkh]] - with D-phenylalanine to 1.25Å
* [[1tkj]] - with D-methionine to 1.15Å
* [[1xbu]] - with ''p''-iodo-D-phenylalanine to 1.20Å

== Historical Context ==

The proteolytic activity contained in the extracellular fluid of cultures of ''Streptomyces griseus'' was first identified by Nomoto and Narahashi (1959a), who obtained a highly purified preparation of this activity from the K-1 strain of this bactreria. A large scale version of their procedure was used to prepare commercial quantities of this preparation (Pronase). Various physical criteria showed that Pronase was homogeneous (Nomoto and Narahashi, 1959b), yet displayed both exopeptidase and endopeptidase activity, with a wide range of side chain specificities (Nomoto and Narahashi, 1959b, 1959c; Nomoto ''et al''., 1960a, 1960b; 1960c). The supposed homogeneity of Pronase was controversial, with other investigators using various chromatographic methods to isolate more fractions with proteolytic activity (Hiramatsu and Ouchi, 1963,; Nomoto et al., 1964). Subsequently Narahashi and Yanagita (1967) identified several distinct proteolytic activities including one which had aminopeptidase activity, and was activated by Ca2+ and Co2+. In contrast to other proteinase activities in the mixture, this aminopeptidase activity displayed considerable heat stablity (up to 80℃) and was unaffected by 9M urea. The activity, was, however, very sensitive to metal chelating agents.

While attempting to isolate the protein responsible for the trypsin activity in Pronase, Vosbeck ''et al.'' (1973) isolated two fractions with aminopeptidase activity. Although the two fractions differed slighty in their molecular weights (23K and 25K), they appeared to have the same enzymatic properties.

Interest in SGAP was renewed when aminopeptidases were recognized as useful tools in assays of metalloendopeptidase activity. The assay was based on a two stage reaction, with the endopeptidase cleaved an N-blocked peptide to release smaller peptide that was rapidly degraded by an aminopeptidase, generating a chromophore (Orlowski and Wilk, 1981; Mumford ''et al''., 1981). SGAP was considered an ideal tool for this purpose, given its stability, small size, and availability (Indig ''et al''., 1990).

== References ==


* Hiramatsu, A., & Ouchi, T. (1963). On the proteolytic enzymes from the commercial protease preparation of ''Streptomyces griseus'' (Pronase P). ''J. Biochem''. '''54''', 462-464.
* Indig, F.E., Benayahu, D., Fried, A., Wientroub, S., Blumberg, S. (1990). Neutral endopeptidase (EC 3.4.24.11) is highly expressed on osteoblastic cells and other marrow stromal cell types. ''Biochem. Biophys. Res. Commun''. '''172''', 620-626. [http://www.ncbi.nlm.nih.gov/pubmed/2241957?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 2241957]
* Mumford, R.A., Pierzchala, P.A., Strauss, A.W., Zimmerman, M. (1981). Purification of a membrane-bound metalloendopeptidase from porcine kidney that degrades peptide hormones. ''Proc. Natl Acad. Sci. USA'' '''78''', 6623-6627. [http://www.ncbi.nlm.nih.gov/pubmed/7031658?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7031658]
* Narahashi, Y., & Yanagita, M. (1967). Studies on proteolytic enzymes (Pronase) of S''treptomyces griseus'' K-1. I. Nature and properties of the proteolytic enzyme system. ''J. Biochem''. (Tokyo) '''62''', 633-641. [http://www.ncbi.nlm.nih.gov/pubmed/4968616?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4968616 ]
* Nomoto, M., & Narahashi, Y. (1959a). A proteolytic enzyme of ''Streptomyces griseus'': I. Purification of a protease of ''Streptomyces grisues''. ''J. Biochem.'' '''46''', 653-667.
* Nomoto, M., & Narahashi, Y. (1959b). A proteolytic enzyme of ''Streptomyces griseus'': III. Homogeneity of the purified enzyme preparation. ''J. Biochem.'' '''46''', 1481-1487.
* Nomoto, M., & Narahashi, Y. (1959c). A proteolytic enzyme of ''Streptomyces griseus'': IV. General properties of ''Streptomyces grisues'' protease. ''J. Biochem.'' '''46''', 1645-1651.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960a). A proteolytic enzyme of ''Streptomyces griseus'': V. Protective effect of calcium ion on the stability of protease. ''J. Biochem.'' '''48''', 453-463.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960b). A proteolytic enzyme of ''Streptomyces griseus'': VI. Hydrolysis of protein by ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 593-602.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960c). A proteolytic enzyme of ''Streptomyces griseus'': VII. Substrate specificity of ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 906-918.
* Nomoto, M., Narahashi, Y., Ouchi, T., & Hiramatsu, A. (1964). Abstract, 6th Inern. Congr. Biochem., N.Y., '''4''', 123.
* Orlowski, M., & Wilk, S. (1981). Purification and specificity of a membrane-bound metalloendopeptidase from bovine pituitaries. ''Biochemistry'' '''20''', 4942-4950. [http://www.ncbi.nlm.nih.gov/pubmed/7028098?ordinalpos=12&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7028098]
* Vosbeck, K. D., Chow, K. F., & Awad, W. M. Jr (1973). The proteolytic enzymes of the K-1 strain of ''Streptomyces griseus'' obtained from a commercial preparation (Pronase). Purification and characterization of the aminopeptidases. '' J. Biol. Chem''. '''248''', 6029-6034. [http://www.ncbi.nlm.nih.gov/pubmed/4199257?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4199257]

--[[User:Harry|Harry]] 15:20, 17 March 2008 (IST)

Streptomyces griseus Aminopeptidase (SGAP)

2008-03-17T12:12:06Z

Harry Greenblatt: /* Structures Available */

Streptomyces griseus Aminopeptidase (SGAP)

2008-03-17T12:03:39Z

Harry Greenblatt: /* Structures Available */

== ''Streptomyces griseus'' Aminopeptidase (SGAP) ==

==Biological function==
''S. griseus'' Aminopeptidase (SGAP; E.C. 3.4.11.-) cleaves the N-terminal amino acid from a peptide or protein, and is specific for larger hydrophobic acids, especially leucine. No cleavage occurs if the next residue is proline.

[[Image:aminopeptidase_rxn2.png | left | thumb| 800px | Reaction catalyzed by SGAP; scissile bond is shown in red.]]
 

== Biological Context ==

SGAP is one of the many proteinases present in the extracellular fluid of cultures of Streptomyces griseus, and can be isolated from [http://en.wikipedia.org/wiki/Pronase Pronase], the commercial preparation of the extracellular fluid from this organism. SGAP is a monomeric, 30KDa, heat stable enzyme requiring two Zn2+ ions for activity, and is activated by Ca2+.

== Structures Available ==

* [[1xjo]] - native SGAP to 1.75Å
* [[1cpj]] - native SGAP to 1.58Å
* [[1qq9]] - SGAP complexed with L-methionine to 1.53Å
* [[1f2o]] - complex with L-leucine to 1.70Å
* [[1f2p]] - complex with L-phenylalanine to 1.80Å

== Historical Context ==

The proteolytic activity contained in the extracellular fluid of cultures of ''Streptomyces griseus'' was first identified by Nomoto and Narahashi (1959a), who obtained a highly purified preparation of this activity from the K-1 strain of this bactreria. A large scale version of their procedure was used to prepare commercial quantities of this preparation (Pronase). Various physical criteria showed that Pronase was homogeneous (Nomoto and Narahashi, 1959b), yet displayed both exopeptidase and endopeptidase activity, with a wide range of side chain specificities (Nomoto and Narahashi, 1959b, 1959c; Nomoto ''et al''., 1960a, 1960b; 1960c). The supposed homogeneity of Pronase was controversial, with other investigators using various chromatographic methods to isolate more fractions with proteolytic activity (Hiramatsu and Ouchi, 1963,; Nomoto et al., 1964). Subsequently Narahashi and Yanagita (1967) identified several distinct proteolytic activities including one which had aminopeptidase activity, and was activated by Ca2+ and Co2+. In contrast to other proteinase activities in the mixture, this aminopeptidase activity displayed considerable heat stablity (up to 80℃) and was unaffected by 9M urea. The activity, was, however, very sensitive to metal chelating agents.

While attempting to isolate the protein responsible for the trypsin activity in Pronase, Vosbeck ''et al.'' (1973) isolated two fractions with aminopeptidase activity. Although the two fractions differed slighty in their molecular weights (23K and 25K), they appeared to have the same enzymatic properties.

Interest in SGAP was renewed when aminopeptidases were recognized as useful tools in assays of metalloendopeptidase activity. The assay was based on a two stage reaction, with the endopeptidase cleaved an N-blocked peptide to release smaller peptide that was rapidly degraded by an aminopeptidase, generating a chromophore (Orlowski and Wilk, 1981; Mumford ''et al''., 1981). SGAP was considered an ideal tool for this purpose, given its stability, small size, and availability (Indig ''et al''., 1990).

== References ==


* Hiramatsu, A., & Ouchi, T. (1963). On the proteolytic enzymes from the commercial protease preparation of ''Streptomyces griseus'' (Pronase P). ''J. Biochem''. '''54''', 462-464.
* Indig, F.E., Benayahu, D., Fried, A., Wientroub, S., Blumberg, S. (1990). Neutral endopeptidase (EC 3.4.24.11) is highly expressed on osteoblastic cells and other marrow stromal cell types. ''Biochem. Biophys. Res. Commun''. '''172''', 620-626. [http://www.ncbi.nlm.nih.gov/pubmed/2241957?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 2241957]
* Mumford, R.A., Pierzchala, P.A., Strauss, A.W., Zimmerman, M. (1981). Purification of a membrane-bound metalloendopeptidase from porcine kidney that degrades peptide hormones. ''Proc. Natl Acad. Sci. USA'' '''78''', 6623-6627. [http://www.ncbi.nlm.nih.gov/pubmed/7031658?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7031658]
* Narahashi, Y., & Yanagita, M. (1967). Studies on proteolytic enzymes (Pronase) of S''treptomyces griseus'' K-1. I. Nature and properties of the proteolytic enzyme system. ''J. Biochem''. (Tokyo) '''62''', 633-641. [http://www.ncbi.nlm.nih.gov/pubmed/4968616?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4968616 ]
* Nomoto, M., & Narahashi, Y. (1959a). A proteolytic enzyme of ''Streptomyces griseus'': I. Purification of a protease of ''Streptomyces grisues''. ''J. Biochem.'' '''46''', 653-667.
* Nomoto, M., & Narahashi, Y. (1959b). A proteolytic enzyme of ''Streptomyces griseus'': III. Homogeneity of the purified enzyme preparation. ''J. Biochem.'' '''46''', 1481-1487.
* Nomoto, M., & Narahashi, Y. (1959c). A proteolytic enzyme of ''Streptomyces griseus'': IV. General properties of ''Streptomyces grisues'' protease. ''J. Biochem.'' '''46''', 1645-1651.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960a). A proteolytic enzyme of ''Streptomyces griseus'': V. Protective effect of calcium ion on the stability of protease. ''J. Biochem.'' '''48''', 453-463.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960b). A proteolytic enzyme of ''Streptomyces griseus'': VI. Hydrolysis of protein by ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 593-602.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960c). A proteolytic enzyme of ''Streptomyces griseus'': VII. Substrate specificity of ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 906-918.
* Nomoto, M., Narahashi, Y., Ouchi, T., & Hiramatsu, A. (1964). Abstract, 6th Inern. Congr. Biochem., N.Y., '''4''', 123.
* Orlowski, M., & Wilk, S. (1981). Purification and specificity of a membrane-bound metalloendopeptidase from bovine pituitaries. ''Biochemistry'' '''20''', 4942-4950. [http://www.ncbi.nlm.nih.gov/pubmed/7028098?ordinalpos=12&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7028098]
* Vosbeck, K. D., Chow, K. F., & Awad, W. M. Jr (1973). The proteolytic enzymes of the K-1 strain of ''Streptomyces griseus'' obtained from a commercial preparation (Pronase). Purification and characterization of the aminopeptidases. '' J. Biol. Chem''. '''248''', 6029-6034. [http://www.ncbi.nlm.nih.gov/pubmed/4199257?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4199257]

Streptomyces griseus Aminopeptidase (SGAP)

2008-03-17T12:00:42Z

Harry Greenblatt: /* Structures Available */

== ''Streptomyces griseus'' Aminopeptidase (SGAP) ==

==Biological function==
''S. griseus'' Aminopeptidase (SGAP; E.C. 3.4.11.-) cleaves the N-terminal amino acid from a peptide or protein, and is specific for larger hydrophobic acids, especially leucine. No cleavage occurs if the next residue is proline.

[[Image:aminopeptidase_rxn2.png | left | thumb| 800px | Reaction catalyzed by SGAP; scissile bond is shown in red.]]
 

== Biological Context ==

SGAP is one of the many proteinases present in the extracellular fluid of cultures of Streptomyces griseus, and can be isolated from [http://en.wikipedia.org/wiki/Pronase Pronase], the commercial preparation of the extracellular fluid from this organism. SGAP is a monomeric, 30KDa, heat stable enzyme requiring two Zn2+ ions for activity, and is activated by Ca2+.

== Structures Available ==

* [[1xjo]] - native SGAP to 1.75Å
* [[1CP7]] - native SGAP to 1.58Å
* [[1QQ9]] - SGAP complexed with L-methionine to 1.53Å
* [[1F2O]] - complex with L-leucine to 1.70Å
* [[1F2P]] - complex with L-phenylalanine to 1.80Å

== Historical Context ==

The proteolytic activity contained in the extracellular fluid of cultures of ''Streptomyces griseus'' was first identified by Nomoto and Narahashi (1959a), who obtained a highly purified preparation of this activity from the K-1 strain of this bactreria. A large scale version of their procedure was used to prepare commercial quantities of this preparation (Pronase). Various physical criteria showed that Pronase was homogeneous (Nomoto and Narahashi, 1959b), yet displayed both exopeptidase and endopeptidase activity, with a wide range of side chain specificities (Nomoto and Narahashi, 1959b, 1959c; Nomoto ''et al''., 1960a, 1960b; 1960c). The supposed homogeneity of Pronase was controversial, with other investigators using various chromatographic methods to isolate more fractions with proteolytic activity (Hiramatsu and Ouchi, 1963,; Nomoto et al., 1964). Subsequently Narahashi and Yanagita (1967) identified several distinct proteolytic activities including one which had aminopeptidase activity, and was activated by Ca2+ and Co2+. In contrast to other proteinase activities in the mixture, this aminopeptidase activity displayed considerable heat stablity (up to 80℃) and was unaffected by 9M urea. The activity, was, however, very sensitive to metal chelating agents.

While attempting to isolate the protein responsible for the trypsin activity in Pronase, Vosbeck ''et al.'' (1973) isolated two fractions with aminopeptidase activity. Although the two fractions differed slighty in their molecular weights (23K and 25K), they appeared to have the same enzymatic properties.

Interest in SGAP was renewed when aminopeptidases were recognized as useful tools in assays of metalloendopeptidase activity. The assay was based on a two stage reaction, with the endopeptidase cleaved an N-blocked peptide to release smaller peptide that was rapidly degraded by an aminopeptidase, generating a chromophore (Orlowski and Wilk, 1981; Mumford ''et al''., 1981). SGAP was considered an ideal tool for this purpose, given its stability, small size, and availability (Indig ''et al''., 1990).

== References ==


* Hiramatsu, A., & Ouchi, T. (1963). On the proteolytic enzymes from the commercial protease preparation of ''Streptomyces griseus'' (Pronase P). ''J. Biochem''. '''54''', 462-464.
* Indig, F.E., Benayahu, D., Fried, A., Wientroub, S., Blumberg, S. (1990). Neutral endopeptidase (EC 3.4.24.11) is highly expressed on osteoblastic cells and other marrow stromal cell types. ''Biochem. Biophys. Res. Commun''. '''172''', 620-626. [http://www.ncbi.nlm.nih.gov/pubmed/2241957?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 2241957]
* Mumford, R.A., Pierzchala, P.A., Strauss, A.W., Zimmerman, M. (1981). Purification of a membrane-bound metalloendopeptidase from porcine kidney that degrades peptide hormones. ''Proc. Natl Acad. Sci. USA'' '''78''', 6623-6627. [http://www.ncbi.nlm.nih.gov/pubmed/7031658?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7031658]
* Narahashi, Y., & Yanagita, M. (1967). Studies on proteolytic enzymes (Pronase) of S''treptomyces griseus'' K-1. I. Nature and properties of the proteolytic enzyme system. ''J. Biochem''. (Tokyo) '''62''', 633-641. [http://www.ncbi.nlm.nih.gov/pubmed/4968616?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4968616 ]
* Nomoto, M., & Narahashi, Y. (1959a). A proteolytic enzyme of ''Streptomyces griseus'': I. Purification of a protease of ''Streptomyces grisues''. ''J. Biochem.'' '''46''', 653-667.
* Nomoto, M., & Narahashi, Y. (1959b). A proteolytic enzyme of ''Streptomyces griseus'': III. Homogeneity of the purified enzyme preparation. ''J. Biochem.'' '''46''', 1481-1487.
* Nomoto, M., & Narahashi, Y. (1959c). A proteolytic enzyme of ''Streptomyces griseus'': IV. General properties of ''Streptomyces grisues'' protease. ''J. Biochem.'' '''46''', 1645-1651.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960a). A proteolytic enzyme of ''Streptomyces griseus'': V. Protective effect of calcium ion on the stability of protease. ''J. Biochem.'' '''48''', 453-463.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960b). A proteolytic enzyme of ''Streptomyces griseus'': VI. Hydrolysis of protein by ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 593-602.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960c). A proteolytic enzyme of ''Streptomyces griseus'': VII. Substrate specificity of ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 906-918.
* Nomoto, M., Narahashi, Y., Ouchi, T., & Hiramatsu, A. (1964). Abstract, 6th Inern. Congr. Biochem., N.Y., '''4''', 123.
* Orlowski, M., & Wilk, S. (1981). Purification and specificity of a membrane-bound metalloendopeptidase from bovine pituitaries. ''Biochemistry'' '''20''', 4942-4950. [http://www.ncbi.nlm.nih.gov/pubmed/7028098?ordinalpos=12&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7028098]
* Vosbeck, K. D., Chow, K. F., & Awad, W. M. Jr (1973). The proteolytic enzymes of the K-1 strain of ''Streptomyces griseus'' obtained from a commercial preparation (Pronase). Purification and characterization of the aminopeptidases. '' J. Biol. Chem''. '''248''', 6029-6034. [http://www.ncbi.nlm.nih.gov/pubmed/4199257?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4199257]

Streptomyces griseus Aminopeptidase (SGAP)

2008-03-17T11:59:48Z

Harry Greenblatt:

== ''Streptomyces griseus'' Aminopeptidase (SGAP) ==

==Biological function==
''S. griseus'' Aminopeptidase (SGAP; E.C. 3.4.11.-) cleaves the N-terminal amino acid from a peptide or protein, and is specific for larger hydrophobic acids, especially leucine. No cleavage occurs if the next residue is proline.

[[Image:aminopeptidase_rxn2.png | left | thumb| 800px | Reaction catalyzed by SGAP; scissile bond is shown in red.]]
 

== Biological Context ==

SGAP is one of the many proteinases present in the extracellular fluid of cultures of Streptomyces griseus, and can be isolated from [http://en.wikipedia.org/wiki/Pronase Pronase], the commercial preparation of the extracellular fluid from this organism. SGAP is a monomeric, 30KDa, heat stable enzyme requiring two Zn2+ ions for activity, and is activated by Ca2+.

== Structures Available ==

* [[1XJO]] - native SGAP to 1.75Å
* [[1CP7]] - native SGAP to 1.58Å
* [[1QQ9]] - SGAP complexed with L-methionine to 1.53Å
* [[1F2O]] - complex with L-leucine to 1.70Å
* [[1F2P]] - complex with L-phenylalanine to 1.80Å

== Historical Context ==

The proteolytic activity contained in the extracellular fluid of cultures of ''Streptomyces griseus'' was first identified by Nomoto and Narahashi (1959a), who obtained a highly purified preparation of this activity from the K-1 strain of this bactreria. A large scale version of their procedure was used to prepare commercial quantities of this preparation (Pronase). Various physical criteria showed that Pronase was homogeneous (Nomoto and Narahashi, 1959b), yet displayed both exopeptidase and endopeptidase activity, with a wide range of side chain specificities (Nomoto and Narahashi, 1959b, 1959c; Nomoto ''et al''., 1960a, 1960b; 1960c). The supposed homogeneity of Pronase was controversial, with other investigators using various chromatographic methods to isolate more fractions with proteolytic activity (Hiramatsu and Ouchi, 1963,; Nomoto et al., 1964). Subsequently Narahashi and Yanagita (1967) identified several distinct proteolytic activities including one which had aminopeptidase activity, and was activated by Ca2+ and Co2+. In contrast to other proteinase activities in the mixture, this aminopeptidase activity displayed considerable heat stablity (up to 80℃) and was unaffected by 9M urea. The activity, was, however, very sensitive to metal chelating agents.

While attempting to isolate the protein responsible for the trypsin activity in Pronase, Vosbeck ''et al.'' (1973) isolated two fractions with aminopeptidase activity. Although the two fractions differed slighty in their molecular weights (23K and 25K), they appeared to have the same enzymatic properties.

Interest in SGAP was renewed when aminopeptidases were recognized as useful tools in assays of metalloendopeptidase activity. The assay was based on a two stage reaction, with the endopeptidase cleaved an N-blocked peptide to release smaller peptide that was rapidly degraded by an aminopeptidase, generating a chromophore (Orlowski and Wilk, 1981; Mumford ''et al''., 1981). SGAP was considered an ideal tool for this purpose, given its stability, small size, and availability (Indig ''et al''., 1990).

== References ==


* Hiramatsu, A., & Ouchi, T. (1963). On the proteolytic enzymes from the commercial protease preparation of ''Streptomyces griseus'' (Pronase P). ''J. Biochem''. '''54''', 462-464.
* Indig, F.E., Benayahu, D., Fried, A., Wientroub, S., Blumberg, S. (1990). Neutral endopeptidase (EC 3.4.24.11) is highly expressed on osteoblastic cells and other marrow stromal cell types. ''Biochem. Biophys. Res. Commun''. '''172''', 620-626. [http://www.ncbi.nlm.nih.gov/pubmed/2241957?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 2241957]
* Mumford, R.A., Pierzchala, P.A., Strauss, A.W., Zimmerman, M. (1981). Purification of a membrane-bound metalloendopeptidase from porcine kidney that degrades peptide hormones. ''Proc. Natl Acad. Sci. USA'' '''78''', 6623-6627. [http://www.ncbi.nlm.nih.gov/pubmed/7031658?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7031658]
* Narahashi, Y., & Yanagita, M. (1967). Studies on proteolytic enzymes (Pronase) of S''treptomyces griseus'' K-1. I. Nature and properties of the proteolytic enzyme system. ''J. Biochem''. (Tokyo) '''62''', 633-641. [http://www.ncbi.nlm.nih.gov/pubmed/4968616?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4968616 ]
* Nomoto, M., & Narahashi, Y. (1959a). A proteolytic enzyme of ''Streptomyces griseus'': I. Purification of a protease of ''Streptomyces grisues''. ''J. Biochem.'' '''46''', 653-667.
* Nomoto, M., & Narahashi, Y. (1959b). A proteolytic enzyme of ''Streptomyces griseus'': III. Homogeneity of the purified enzyme preparation. ''J. Biochem.'' '''46''', 1481-1487.
* Nomoto, M., & Narahashi, Y. (1959c). A proteolytic enzyme of ''Streptomyces griseus'': IV. General properties of ''Streptomyces grisues'' protease. ''J. Biochem.'' '''46''', 1645-1651.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960a). A proteolytic enzyme of ''Streptomyces griseus'': V. Protective effect of calcium ion on the stability of protease. ''J. Biochem.'' '''48''', 453-463.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960b). A proteolytic enzyme of ''Streptomyces griseus'': VI. Hydrolysis of protein by ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 593-602.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960c). A proteolytic enzyme of ''Streptomyces griseus'': VII. Substrate specificity of ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 906-918.
* Nomoto, M., Narahashi, Y., Ouchi, T., & Hiramatsu, A. (1964). Abstract, 6th Inern. Congr. Biochem., N.Y., '''4''', 123.
* Orlowski, M., & Wilk, S. (1981). Purification and specificity of a membrane-bound metalloendopeptidase from bovine pituitaries. ''Biochemistry'' '''20''', 4942-4950. [http://www.ncbi.nlm.nih.gov/pubmed/7028098?ordinalpos=12&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7028098]
* Vosbeck, K. D., Chow, K. F., & Awad, W. M. Jr (1973). The proteolytic enzymes of the K-1 strain of ''Streptomyces griseus'' obtained from a commercial preparation (Pronase). Purification and characterization of the aminopeptidases. '' J. Biol. Chem''. '''248''', 6029-6034. [http://www.ncbi.nlm.nih.gov/pubmed/4199257?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4199257]

Streptomyces griseus Aminopeptidase (SGAP)

2008-03-17T11:30:07Z

Harry Greenblatt:

== ''Streptomyces griseus'' Aminopeptidase (SGAP) ==

==Biological function==
''S. griseus'' Aminopeptidase (SGAP; E.C. 3.4.11.-) cleaves the N-terminal amino acid from a peptide or protein, and is specific for larger hydrophobic acids, especially leucine. No cleavage occurs if the next residue is proline.

[[Image:aminopeptidase_rxn2.png | left | thumb| 800px | Reaction catalyzed by SGAP; scissile bond is shown in red.]]
 

== Biological Context ==

SGAP is one of the many proteinases present in the extracellular fluid of cultures of Streptomyces griseus, and can be isolated from [http://en.wikipedia.org/wiki/Pronase Pronase], the commercial preparation of the extracellular fluid from this organism. SGAP is a monomeric, 30KDa, heat stable enzyme requiring two Zn2+ ions for activity, and is activated by Ca2+.

== Historical Context ==

The proteolytic activity contained in the extracellular fluid of cultures of ''Streptomyces griseus'' was first identified by Nomoto and Narahashi (1959a), who obtained a highly purified preparation of this activity from the K-1 strain of this bactreria. A large scale version of their procedure was used to prepare commercial quantities of this preparation (Pronase). Various physical criteria showed that Pronase was homogeneous (Nomoto and Narahashi, 1959b), yet displayed both exopeptidase and endopeptidase activity, with a wide range of side chain specificities (Nomoto and Narahashi, 1959b, 1959c; Nomoto ''et al''., 1960a, 1960b; 1960c). The supposed homogeneity of Pronase was controversial, with other investigators using various chromatographic methods to isolate more fractions with proteolytic activity (Hiramatsu and Ouchi, 1963,; Nomoto et al., 1964). Subsequently Narahashi and Yanagita (1967) identified several distinct proteolytic activities including one which had aminopeptidase activity, and was activated by Ca2+ and Co2+. In contrast to other proteinase activities in the mixture, this aminopeptidase activity displayed considerable heat stablity (up to 80℃) and was unaffected by 9M urea. The activity, was, however, very sensitive to metal chelating agents.

While attempting to isolate the protein responsible for the trypsin activity in Pronase, Vosbeck ''et al.'' (1973) isolated two fractions with aminopeptidase activity. Although the two fractions differed slighty in their molecular weights (23K and 25K), they appeared to have the same enzymatic properties.

Interest in SGAP was renewed when aminopeptidases were recognized as useful tools in assays of metalloendopeptidase activity. The assay was based on a two stage reaction, with the endopeptidase cleaved an N-blocked peptide to release smaller peptide that was rapidly degraded by an aminopeptidase, generating a chromophore (Orlowski and Wilk, 1981; Mumford ''et al''., 1981). SGAP was considered an ideal tool for this purpose, given its stability, small size, and availability (Indig ''et al''., 1990).

== References ==


* Hiramatsu, A., & Ouchi, T. (1963). On the proteolytic enzymes from the commercial protease preparation of ''Streptomyces griseus'' (Pronase P). ''J. Biochem''. '''54''', 462-464.
* Indig, F.E., Benayahu, D., Fried, A., Wientroub, S., Blumberg, S. (1990). Neutral endopeptidase (EC 3.4.24.11) is highly expressed on osteoblastic cells and other marrow stromal cell types. ''Biochem. Biophys. Res. Commun''. '''172''', 620-626. [http://www.ncbi.nlm.nih.gov/pubmed/2241957?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 2241957]
* Mumford, R.A., Pierzchala, P.A., Strauss, A.W., Zimmerman, M. (1981). Purification of a membrane-bound metalloendopeptidase from porcine kidney that degrades peptide hormones. ''Proc. Natl Acad. Sci. USA'' '''78''', 6623-6627. [http://www.ncbi.nlm.nih.gov/pubmed/7031658?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7031658]
* Narahashi, Y., & Yanagita, M. (1967). Studies on proteolytic enzymes (Pronase) of S''treptomyces griseus'' K-1. I. Nature and properties of the proteolytic enzyme system. ''J. Biochem''. (Tokyo) '''62''', 633-641. [http://www.ncbi.nlm.nih.gov/pubmed/4968616?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4968616 ]
* Nomoto, M., & Narahashi, Y. (1959a). A proteolytic enzyme of ''Streptomyces griseus'': I. Purification of a protease of ''Streptomyces grisues''. ''J. Biochem.'' '''46''', 653-667.
* Nomoto, M., & Narahashi, Y. (1959b). A proteolytic enzyme of ''Streptomyces griseus'': III. Homogeneity of the purified enzyme preparation. ''J. Biochem.'' '''46''', 1481-1487.
* Nomoto, M., & Narahashi, Y. (1959c). A proteolytic enzyme of ''Streptomyces griseus'': IV. General properties of ''Streptomyces grisues'' protease. ''J. Biochem.'' '''46''', 1645-1651.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960a). A proteolytic enzyme of ''Streptomyces griseus'': V. Protective effect of calcium ion on the stability of protease. ''J. Biochem.'' '''48''', 453-463.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960b). A proteolytic enzyme of ''Streptomyces griseus'': VI. Hydrolysis of protein by ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 593-602.
* Nomoto, M., & Narahashi, Y., Murakami, M. (1960c). A proteolytic enzyme of ''Streptomyces griseus'': VII. Substrate specificity of ''Streptomyces griseus'' protease. ''J. Biochem.'' '''48''', 906-918.
* Nomoto, M., Narahashi, Y., Ouchi, T., & Hiramatsu, A. (1964). Abstract, 6th Inern. Congr. Biochem., N.Y., '''4''', 123.
* Orlowski, M., & Wilk, S. (1981). Purification and specificity of a membrane-bound metalloendopeptidase from bovine pituitaries. ''Biochemistry'' '''20''', 4942-4950. [http://www.ncbi.nlm.nih.gov/pubmed/7028098?ordinalpos=12&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 7028098]
* Vosbeck, K. D., Chow, K. F., & Awad, W. M. Jr (1973). The proteolytic enzymes of the K-1 strain of ''Streptomyces griseus'' obtained from a commercial preparation (Pronase). Purification and characterization of the aminopeptidases. '' J. Biol. Chem''. '''248''', 6029-6034. [http://www.ncbi.nlm.nih.gov/pubmed/4199257?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum PMID: 4199257]