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Oxyanion and tetrahedral intermediate stabilization by subtilisin : detection of a new tetrahedral adduct
Howe, Nicole; Rogers, Louis; Hewage, Chandralal; Malthouse, J.Paul G.
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. Science Foundation Ireland Higher Education Authority
Keyword(s): Subtilisin; Glyoxal inhibitor; Protease; Chymotrypsin; pH; Oxyanion; Proteolytic enzymes; Hydrogen-ion concentration; Subtilisins; Peptide Hydrolases; Hydrogen-Ion Concentration
Publication Date:
Type: Journal article
Peer-Reviewed: Unknown
Language(s): English
Institution: University College Dublin
Publisher(s): Elsevier
File Format(s): other; application/pdf
First Indexed: 2012-08-25 05:18:01 Last Updated: 2018-10-11 16:32:49