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

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dc.contributor.author Howe, Nicole
dc.contributor.author Rogers, Louis
dc.contributor.author Hewage, Chandralal
dc.contributor.author Malthouse, J.Paul G.
dc.date.accessioned 2011-07-19T14:24:39Z
dc.date.available 2011-07-19T14:24:39Z
dc.date.copyright 2009 Elsevier B.V. en
dc.date.issued 2009-08
dc.identifier.citation Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics en
dc.identifier.issn 1570-9639
dc.identifier.uri http://hdl.handle.net/10197/3022
dc.description.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. en
dc.description.sponsorship Science Foundation Ireland en
dc.description.sponsorship Higher Education Authority en
dc.format.extent 1435848 bytes
dc.format.mimetype application/pdf
dc.language.iso en en
dc.publisher Elsevier en
dc.rights This is the author’s version of a work that was accepted for publication in Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics). Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in BBA - Proteins and Proteomics, Volume 1794, Issue 8, pages 1251-1258, DOI: 10.1016/j.bbapap.2009.04.007 en
dc.subject Subtilisin en
dc.subject Glyoxal inhibitor en
dc.subject Protease en
dc.subject Chymotrypsin en
dc.subject pH en
dc.subject Oxyanion en
dc.subject.lcsh Proteolytic enzymes en
dc.subject.lcsh Chymotrypsin en
dc.subject.lcsh Hydrogen-ion concentration en
dc.subject.lcsh Subtilisins en
dc.subject.mesh Peptide Hydrolases en
dc.subject.mesh Chymotrypsin en
dc.subject.mesh Hydrogen-Ion Concentration en
dc.subject.mesh Subtilisin en
dc.title Oxyanion and tetrahedral intermediate stabilization by subtilisin : detection of a new tetrahedral adduct en
dc.type Journal Article en
dc.internal.availability Full text available en
dc.internal.webversions Publisher's version en
dc.internal.webversions http://dx.doi.org/10.1016/j.bbapap.2009.04.007 en
dc.status Peer reviewed en
dc.identifier.volume 1794 en
dc.identifier.issue 8 en
dc.identifier.startpage 1251 en
dc.identifier.endpage 1258 en
dc.identifier.doi 10.1016/j.bbapap.2009.04.007
dc.neeo.contributor Howe|Nicole|aut| en
dc.neeo.contributor Rogers|Louis|aut| en
dc.neeo.contributor Hewage|Chandralal|aut| en
dc.neeo.contributor Malthouse|J.Paul G.|aut| en


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