title: Solid-State17O NMR of Unstable Acyl-Enzyme Intermediates: A Direct Probe of Hydrogen Bonding Interactions in the Oxyanion Hole of Serine Proteases.
abstract: We report preparation, trapping, and solid-state17O NMR characterization of three unstable acyl-enzyme intermediates (≈ 26 kDa): p-N,N-dimethylamino-[17O]benzoyl-chymotrypsin, trans-o-methoxy-[17O]cinnamoyl-chymotrypsin, and trans-p-methoxy-[17O]cinnamoyl-chymotrypsin. We show that both the17O chemical shifts and nuclear quadrupolar parameters obtained for these acyl-enzyme intermediates in the solid state are correlated with their deacylation rate constants measured in aqueous solution. With the aid of quantum mechanical calculations, the experimental17O NMR parameters were interpreted as to reflect the hydrogen bonding interactions between the carbonyl (C═17O) functional group of the acyl moiety and the two NH groups from the protein backbone (Ser195 and Gly193) in the oxyanion hole, a general feature of all serine proteases. Our results further suggest that the17O chemical shift and quadrupole coupling constant display distinctly different sensitivities toward different aspects of hydrogen bonding, such as hydrogen bond distance and direction. This work demonstrates the utility of17O as a useful nuclear probe in NMR studies of enzymes.
authors.affiliation: Department of Chemistry, Queen's University , 90 Bader Lane, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Aaron W
authors.initials: AW
authors.lastname: Tang
authors.affiliation: Department of Chemistry, Queen's University , 90 Bader Lane, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Xianqi
authors.initials: X
authors.lastname: Kong
authors.affiliation: Department of Chemistry, Queen's University , 90 Bader Lane, Kingston, Ontario K7L 3N6, Canada.|Department of Chemistry, University of Ottawa , Ottawa, Ontario K1N 6N5, Canada.
authors.forename: Victor
authors.initials: V
authors.lastname: Terskikh
authors.affiliation: Department of Chemistry, Queen's University , 90 Bader Lane, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Gang
authors.initials: G
authors.lastname: Wu
mesh_terms: D000838:Anions / Q000737:chemistry; D002918:Chymotrypsin / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D010100:Oxygen / Q000737:chemistry*; D010103:Oxygen Isotopes; D057057:Serine Proteases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000838:Anions; D010103:Oxygen Isotopes; D057057:Serine Proteases; D002918:Chymotrypsin; D010100:Oxygen