MECHANISM OF ACTION OF DD-PEPTIDASES - ROLE OF ASPARAGINE-161 IN THE STREPTOMYCES R61 DD-PEPTIDASE

The role of residue Asn-161 in the interaction between the Steptomyces R61 DD-peptidase and various substrates or beta-lactam inactivators w as probed by site-directed mutagenesis. The residue was successively r eplaced by serine and alanine. In the first case, acylation rates were mainly affected with the peptide and ester substrates but not with th e thiol-ester substrates and beta-lactams. However, the deacylation ra tes were decreased 10-30-fold with the substrates yielding benzoylglyc yl 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. Surprisi ngly, the deacylation rates of the benzoylglycyl and benzoylalanyl add ucts 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 compa red with those obtained with the Asn-132 mutants of a class A beta-lac tamase.