Oxyanion and tetrahedral intermediate stabilization by subtilisin : detection of a new tetrahedral adduct

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Title: Oxyanion and tetrahedral intermediate stabilization by subtilisin : detection of a new tetrahedral adduct
Authors: Howe, NicoleRogers, LouisHewage, ChandralalMalthouse, J.Paul G.
Permanent link: http://hdl.handle.net/10197/3022
Date: Aug-2009
Online since: 2011-07-19T14:24:39Z
Abstract: The peptide-derived glyoxal inhibitor Z-Ala-Ala-Phe-glyoxal has been shown to be ~10 fold more effective as an inhibitor of subtilisin than Z-Ala-Pro-Phe-glyoxal. Signals at 107.2 p.p.m. and 200.5 p.p.m. are observed for the glyoxal keto and aldehyde carbons of the inhibitor bound to subtilisin, showing that the glyoxal keto and aldehyde carbons are sp3 and sp2 hybridized respectively. The signal at 107.2 p.p.m. from the carbon atom attached to the hemiketal oxyanion is formed in a slow exchange process that involves the dehydration of the glyoxal aldehyde carbon. Two additional signals are observed one at 108.2 p.p.m. and the other at 90.9 p.p.m. for the glyoxal keto and aldehyde carbons respectively at pHs 6-8 demonstrating that subtilisin forms an additional tetrahedral adduct with Z-Ala-Ala-Phe-glyoxal in which both the glyoxal keto and aldehyde carbons are sp3 hybridised. For the first time we can quantify oxyanion stabilisation in subtilisin. We conclude that oxyanion stabilisation is more effective in subtilisin than in chymotrypsin. Using 1H-NMR we show that the binding of Z-Ala-Ala-Phe-glyoxal to subtilisin raises the pKa of the imidazolium ion of the active site histidine residue promoting oxyanion stabilisation. The mechanistic significance of these results are discussed.
Funding Details: Science Foundation Ireland
Higher Education Authority
Type of material: Journal Article
Publisher: Elsevier
Journal: Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics
Volume: 1794
Issue: 8
Start page: 1251
End page: 1258
Copyright (published version): 2009 Elsevier B.V.
Keywords: SubtilisinGlyoxal inhibitorProteaseChymotrypsinpHOxyanion
Subject LCSH: Proteolytic enzymes
Chymotrypsin
Hydrogen-ion concentration
Subtilisins
DOI: 10.1016/j.bbapap.2009.04.007
Other versions: http://dx.doi.org/10.1016/j.bbapap.2009.04.007
Language: en
Status of Item: Peer reviewed
ISSN: 1570-9639
This item is made available under a Creative Commons License: https://creativecommons.org/licenses/by-nc-sa/1.0/
Appears in Collections:Biomolecular and Biomedical Science Research Collection

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