title: Importance of hydrogen-bonding interactions involving the side chain of Asp158 in the catalytic mechanism of papain.
abstract: In a previous study, it was shown that replacing Asp158 in papain by Asn had little effect on activity and that the negatively charged carboxylate of Asp158 does not significantly stabilize the active site thiolate-imidazolium ion pair of papain (Ménard et al., 1990). In this paper, we report the kinetic characterization of three more mutants at this position: Asp158Gly, Asp158Ala, and Asp158Glu. From the pH-activity profiles of these and other mutants of papain, it has been possible to develop a model that enables us to dissect out the contribution of the various mutations toward (i) intrinsic activity, (ii) ion pair stability, and (iii) the electrostatic potential at the active site. Results obtained with mutants that place either Gly or Ala at position 158 indicate that the hydrogen bonds involving the side chain of Asp158 in wild-type papain are indirectly important for enzyme activity. When CBZ-Phe-Arg-MCA is used as a substrate, the (kcat/KM)obs values at pH 6.5 are 3650 and 494 M-1 s-1 for Asp158Gly and Asp158Ala, respectively, as compared to 119,000 M-1 s-1 for papain. Results with the Asp158Glu mutant suggest that the side chain of Glu moves closer to the active site and cannot form hydrogen bonds similar to those involving Asp158 in papain. From the four mutations introduced at position 158 in papain, we can conclude that it is not the charge but the hydrogen-bonding interactions involving the side chain of Asp158 that contribute the most to the stabilization of the thiolate-imidazolium ion pair in papain. However, the charge and the hydrogen bonds of Asp158 both contribute to the intrinsic activity of the enzyme.
authors.affiliation: Protein Engineering Section, National Research Council of Canada, Montréal, Québec.
authors.forename: R
authors.initials: R
authors.lastname: Ménard
authors.forename: H E
authors.initials: HE
authors.lastname: Khouri
authors.forename: C
authors.initials: C
authors.lastname: Plouffe
authors.forename: P
authors.initials: P
authors.lastname: Laflamme
authors.forename: R
authors.initials: R
authors.lastname: Dupras
authors.forename: T
authors.initials: T
authors.lastname: Vernet
authors.forename: D C
authors.initials: DC
authors.lastname: Tessier
authors.forename: D Y
authors.initials: DY
authors.lastname: Thomas
authors.forename: A C
authors.initials: AC
authors.lastname: Storer
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010206:Papain / Q000737:chemistry* / Q000235:genetics
chemical_list: D009838:Oligodeoxyribonucleotides; D001224:Aspartic Acid; D010206:Papain