title: Oxyanion and tetrahedral intermediate stabilisation by subtilisin: detection of a new tetrahedral adduct.
abstract: The peptide-derived glyoxal inhibitor Z-Ala-Ala-Phe-glyoxal has been shown to be approximately 10 fold more effective as an inhibitor of subtilisin than Z-Ala-Pro-Phe-glyoxal. Signals at 107.2 ppm and 200.5 ppm are observed for the glyoxal keto and aldehyde carbons of the inhibitor bound to subtilisin, showing that the glyoxal keto and aldehyde carbons are sp(3) and sp(2) hybridised respectively. The signal at 107.2 ppm 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 ppm and the other at 90.9 ppm 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 sp(3) 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 (1)H-NMR we show that the binding of Z-Ala-Ala-Phe-glyoxal to subtilisin raises the pK(a) of the imidazolium ion of the active site histidine residue promoting oxyanion stabilisation. The mechanistic significance of these results is discussed.
authors.affiliation: UCD School of Biomolecular and Biomedical Science, UCD Centre for Synthesis and Chemical Biology, SEC Strategic Research Cluster, Conway Institute, University College Dublin, Dublin 4, Ireland.
authors.forename: Nicole
authors.initials: N
authors.lastname: Howe
authors.forename: Louis
authors.initials: L
authors.lastname: Rogers
authors.forename: Chandralal
authors.initials: C
authors.lastname: Hewage
authors.forename: J Paul G
authors.initials: JP
authors.lastname: Malthouse
mesh_terms: D000838:Anions / Q000737:chemistry*; D002247:Carbon Isotopes; D006037:Glyoxal / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D019906:Nuclear Magnetic Resonance, Biomolecular; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology*; D012697:Serine Endopeptidases / Q000378:metabolism; D020860:Subtilisin / Q000378:metabolism*; D013381:Subtilisins / Q000037:antagonists & inhibitors
chemical_list: D000838:Anions; D002247:Carbon Isotopes; D009842:Oligopeptides; C501834:Z-Ala-Pro-Phe-glyoxal; C543106:benzyloxycarbonyl-alanyl-alanyl-phenylalanyl-glyoxal; D006037:Glyoxal; D012697:Serine Endopeptidases; D013381:Subtilisins; D020860:Subtilisin