1scn
From Proteopedia
(New page: 200px<br /><applet load="1scn" size="450" color="white" frame="true" align="right" spinBox="true" caption="1scn, resolution 1.9Å" /> '''INACTIVATION OF SUBTI...) |
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| - | [[Image:1scn.jpg|left|200px]]<br /><applet load="1scn" size=" | + | [[Image:1scn.jpg|left|200px]]<br /><applet load="1scn" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1scn, resolution 1.9Å" /> | caption="1scn, resolution 1.9Å" /> | ||
'''INACTIVATION OF SUBTILISIN CARLSBERG BY N-(TERT-BUTOXYCARBONYL-ALANYL-PROLYL-PHENYLALANYL)-O-BENZOYL HYDROXYLAMINE: FORMATION OF COVALENT ENZYME-INHIBITOR LINKAGE IN THE FORM OF A CARBAMATE DERIVATIVE'''<br /> | '''INACTIVATION OF SUBTILISIN CARLSBERG BY N-(TERT-BUTOXYCARBONYL-ALANYL-PROLYL-PHENYLALANYL)-O-BENZOYL HYDROXYLAMINE: FORMATION OF COVALENT ENZYME-INHIBITOR LINKAGE IN THE FORM OF A CARBAMATE DERIVATIVE'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The mechanism of inactivation of serine proteases by | + | The mechanism of inactivation of serine proteases by N-peptidyl-O-aroylhydroxylamines was studied by X-ray crystallography. Cocrystals of subtilisin Carlsberg inactivated with N-((tert-butoxycarbonyl)alanylprolylphenylalanyl)-O-nitrobenzoy lhydroxylamine were grown, and diffraction data to 1.8-A resolution were obtained. The resulting electron density maps clearly reveal that the gamma-oxygen of the catalytic serine forms a carbamate derivative with the inhibitor. The peptide part of the inhibitor does not form the usual antiparallel beta-sheet in the P binding cleft but protrudes out of the active site and is stabilized by a network of water molecules. These results, combined with kinetic characterization reported previously [Demuth, H.-U., Schoenlein, C., & Barth, A. (1989b) Biochim. Biophys. Acta 996, 19-22; Schmidt, C., Schmidt, R., & Demuth, H.-U. (1990) Peptides (Giralt, E., & Andreu, D., Eds.) ESCOM Science Publishers B.V., Amsterdam] support the existence of at least one intermediate between the formation of the Michaelis complex and the final product. We suggest a mechanism for the inactivation of subtilisin Carlsberg by N-((tert-butoxycarbonyl)alanylprolylphenylalanyl)-O-benzoylhydr oxylamine whereby a negatively charged Michaelis complex undergoes a Lossen rearrangement giving rise to an isocyanate intermediate that reacts with the side chain of the active site serine. |
==About this Structure== | ==About this Structure== | ||
| - | 1SCN is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/ ] with CA and NA as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Subtilisin Subtilisin], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.4.21.62 3.4.21.62] Full crystallographic information is available from [http:// | + | 1SCN is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/ ] with <scene name='pdbligand=CA:'>CA</scene> and <scene name='pdbligand=NA:'>NA</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Subtilisin Subtilisin], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.4.21.62 3.4.21.62] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1SCN OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Subtilisin]] | [[Category: Subtilisin]] | ||
| - | [[Category: Demuth, H | + | [[Category: Demuth, H U.]] |
[[Category: Ringe, D.]] | [[Category: Ringe, D.]] | ||
| - | [[Category: Steinmetz, A | + | [[Category: Steinmetz, A C.U.]] |
[[Category: CA]] | [[Category: CA]] | ||
[[Category: NA]] | [[Category: NA]] | ||
[[Category: complex(serine proteinase/inhibitor)]] | [[Category: complex(serine proteinase/inhibitor)]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 15:00:11 2008'' |
Revision as of 13:00, 21 February 2008
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INACTIVATION OF SUBTILISIN CARLSBERG BY N-(TERT-BUTOXYCARBONYL-ALANYL-PROLYL-PHENYLALANYL)-O-BENZOYL HYDROXYLAMINE: FORMATION OF COVALENT ENZYME-INHIBITOR LINKAGE IN THE FORM OF A CARBAMATE DERIVATIVE
Overview
The mechanism of inactivation of serine proteases by N-peptidyl-O-aroylhydroxylamines was studied by X-ray crystallography. Cocrystals of subtilisin Carlsberg inactivated with N-((tert-butoxycarbonyl)alanylprolylphenylalanyl)-O-nitrobenzoy lhydroxylamine were grown, and diffraction data to 1.8-A resolution were obtained. The resulting electron density maps clearly reveal that the gamma-oxygen of the catalytic serine forms a carbamate derivative with the inhibitor. The peptide part of the inhibitor does not form the usual antiparallel beta-sheet in the P binding cleft but protrudes out of the active site and is stabilized by a network of water molecules. These results, combined with kinetic characterization reported previously [Demuth, H.-U., Schoenlein, C., & Barth, A. (1989b) Biochim. Biophys. Acta 996, 19-22; Schmidt, C., Schmidt, R., & Demuth, H.-U. (1990) Peptides (Giralt, E., & Andreu, D., Eds.) ESCOM Science Publishers B.V., Amsterdam] support the existence of at least one intermediate between the formation of the Michaelis complex and the final product. We suggest a mechanism for the inactivation of subtilisin Carlsberg by N-((tert-butoxycarbonyl)alanylprolylphenylalanyl)-O-benzoylhydr oxylamine whereby a negatively charged Michaelis complex undergoes a Lossen rearrangement giving rise to an isocyanate intermediate that reacts with the side chain of the active site serine.
About this Structure
1SCN is a Single protein structure of sequence from [1] with and as ligands. Active as Subtilisin, with EC number 3.4.21.62 Full crystallographic information is available from OCA.
Reference
Inactivation of subtilisin Carlsberg by N-((tert-butoxycarbonyl)alanylprolylphenylalanyl)-O-benzoylhydroxyl- amine: formation of a covalent enzyme-inhibitor linkage in the form of a carbamate derivative., Steinmetz AC, Demuth HU, Ringe D, Biochemistry. 1994 Aug 30;33(34):10535-44. PMID:8068694
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