title: Determination of the structure of tetrahedral transition state analogues bound at the active site of chymotrypsin using 18O and 2H isotope shifts in the 13C NMR spectra of glyoxal inhibitors.
abstract: The peptide-derived glyoxal inhibitor Z-Ala-Pro-Phe-glyoxal, where Z is benzyloxycarbonyl, is an extremely potent inhibitor of chymotrypsin. When it is bound to chymotrypsin both the glyoxal (RCOCHO) keto and aldehyde carbons are sp3 hybridized with chemical shifts of 100.7 and 91.4 ppm, respectively. However it is has not been shown whether these carbons are bound as hydrates or whether the active-site serine has reacted with them to form the corresponding hemiketal or hemiacetal. In this study we use 18O isotope shifts to determine whether one or two exchangeable oxygen atoms are attached to the glyoxal keto or aldehyde carbons when it is free in water or bound to alpha-chymotrypsin. Both the 18O isotope shifts at the free and enzyme-bound aldehyde carbons were approximately 0.04 ppm showing that it is hydrated in both the free and bound forms. The 18O isotope shift for the free hydrated keto carbon at 96.6 ppm was 0.046-0.049 ppm, but this was reduced to 0.026 ppm when the glyoxal inhibitor was bound to alpha-chymotrypsin showing that the nonexchangeable serine hydroxyl group has formed a hemiketal with glyoxal keto carbon. Deuterium isotope shifts on the 13C NMR signals from the glyoxal inhibitor when it free and hydrated, when it is bound to chymotrypsin, as well as when it forms a model hemiketal confirm that the serine hydroxyl group has formed a hemiketal with the glyoxal keto carbon. The reasons for the different reaction specificities of glyoxal inhibitors for the active-site nucleophiles of serine and cysteine proteases are discussed.
authors.affiliation: UCD School of Biomolecular and Biomedical Science, Centre for Synthesis and Chemical Biology, UCD Conway Institute, University College Dublin, Dublin 4, Ireland.
authors.forename: Edward
authors.initials: E
authors.lastname: Spink
authors.forename: Chandralal
authors.initials: C
authors.lastname: Hewage
authors.forename: J Paul G
authors.initials: JP
authors.lastname: Malthouse
mesh_terms: D001665:Binding Sites; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry*; D041961:Deuterium Exchange Measurement; D006037:Glyoxal / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D008954:Models, Biological; D019906:Nuclear Magnetic Resonance, Biomolecular; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D010103:Oxygen Isotopes / Q000737:chemistry*; D011485:Protein Binding
chemical_list: D009842:Oligopeptides; D010103:Oxygen Isotopes; C501834:Z-Ala-Pro-Phe-glyoxal; D006037:Glyoxal; D003903:Deuterium; D002918:Chymotrypsin