title: Removal of an inter-domain hydrogen bond through site-directed mutagenesis: role of serine 176 in the mechanism of papain.
abstract: A mutant of papain, where an inter-domain hydrogen bond between the side chain hydroxyl group of a serine residue at position 176 and the side chain carbonyl oxygen of a glutamine residue at position 19 has been removed by site-directed mutagenesis, has been produced and characterized kinetically. The mutation of Ser176 to an alanine has only a small effect on the kinetic parameters, the kcat/Km for hydrolysis of CBZ-Phe-Arg-MCA by the Ser176Ala enzyme being of 8.1 x 10(4) /M/s compared with 1.2 x 10(5) /M/s for papain. Serine 176 is therefore not essential for the catalytic functioning of papain, even though this residue is conserved in all cysteine proteases sequenced. The pH-activity profiles were shown to be narrower in the mutant enzyme by up to 1 pH unit at high ionic strength. This result is interpreted to indicate that replacing Ser176 by an alanine destabilizes the thiolate-imidazolium form of the catalytic site Cys25-His159 residues of papain. Possible explanations for that effect are given and the role of a serine residue at position 176 in papain is discussed.
authors.affiliation: Protein Engineering Section, National Research Council Canada, Montréal.
authors.forename: R
authors.initials: R
authors.lastname: Ménard
authors.forename: C
authors.initials: C
authors.lastname: Plouffe
authors.forename: H E
authors.initials: HE
authors.lastname: Khouri
authors.forename: R
authors.initials: R
authors.lastname: Dupras
authors.forename: D C
authors.initials: DC
authors.lastname: Tessier
authors.forename: T
authors.initials: T
authors.lastname: Vernet
authors.forename: D Y
authors.initials: DY
authors.lastname: Thomas
authors.forename: A C
authors.initials: AC
authors.lastname: Storer
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010206:Papain / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D012694:Serine / Q000737:chemistry*
chemical_list: D015345:Oligonucleotide Probes; D012694:Serine; D010206:Papain