title: Mechanism of action of DD-peptidases: role of asparagine-161 in the Streptomyces R61 DD-peptidase.
abstract: The role of residue Asn-161 in the interaction between the Streptomyces R61 DD-peptidase and various substrates or beta-lactam inactivators was probed by site-directed mutagenesis. The residue was successively replaced by serine and alanine. In the first case, acylation rates were mainly affected with the peptide and ester substrates but not with the thiol-ester substrates and beta-lactams. However, the deacylation rates were decreased 10-30-fold with the substrates yielding benzoylglycyl and benzoylalanyl adducts. The Asn161Ala mutant was more generally affected, although the acylation rates with cefuroxime and cefotaxime remained similar to those observed with the wild-type enzyme. Surprisingly, the deacylation rates of the benzoylglycyl and benzoylalanyl adducts were very close to those observed with the wild-type enzyme. The results also indicate that the interaction with the peptide substrate and the transpeptidation reaction were more sensitive to the mutations than the other reactions studied. The results are discussed and compared with those obtained with the Asn-132 mutants of a class A beta-lactamase.
authors.affiliation: Centre d'Ingéniérie des Protéines, Université de Liége, Belgium.
authors.forename: J M
authors.initials: JM
authors.lastname: Wilkin
authors.forename: M
authors.initials: M
authors.lastname: Jamin
authors.forename: B
authors.initials: B
authors.lastname: Joris
authors.forename: J M
authors.initials: JM
authors.lastname: Frere
mesh_terms: D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002268:Carboxypeptidases / Q000737:chemistry / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D001216:Asparagine; D002268:Carboxypeptidases; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase