Oxyanion and tetrahedral intermediate stabilization by subtilisin : detection of a new tetrahedral adduct
|Title:||Oxyanion and tetrahedral intermediate stabilization by subtilisin : detection of a new tetrahedral adduct||Authors:||Howe, Nicole
Malthouse, J.Paul G.
|Permanent link:||http://hdl.handle.net/10197/3022||Date:||Aug-2009||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:||Subtilisin; Glyoxal inhibitor; Protease; Chymotrypsin; pH; Oxyanion||Subject LCSH:||Proteolytic enzymes
|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|
|Appears in Collections:||Biomolecular and Biomedical Science Research Collection|
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