ROLE OF RESIDUE LYS315 IN THE MECHANISM OF ACTION OF THE ENTEROBACTER-CLOACAE 908R BETA-LACTAMASE

The role of the highly conserved Lys315 residue in the catalytic mecha nism of a class C beta-lactamase has been probed by site-directed muta genesis. Lys315 has been replaced by a histidine in the Enterobacter c loacae 908R beta-lactamase, thus introducing a tritratabre group to pr obe the role of the positive charge, and by a glutamine. The effects o f these mutations have been studied on the kinetics of penicillin G an d cephalothin turnover and on the pre-steady-state kinetics with carbe nicillin at different pH. Results showed that substrate binding was no t impaired by the mutations, so that an interaction with the substrate -free carboxylate in the Henri-Michaelis complex could be ruled out. L ys315 must have a catalytic role as shown by the decreased acylation a nd deacylation rates observed with the mutant enzymes. The mutants exh ibited a lower activity at acidic pH, and this observation could be co rrelated with a decreased affinity for (3-aminophenyl)boronate, a comp ound devoid of free carboxylate which binds to the active site and for ms an adduct mimicking the tetrahedral intermediate. This suggested th at Lys315 was somehow involved in accelerating the nucleophilic substi tutions along the reaction pathway. The study was extended to modified substrates where the free carboxylate had been esterified. Neither ac ylation nor deacylation seemed severely impaired with these compounds, showing that the interaction between the enzyme and the substrate-fre e carboxylate did not play a major role in catalysis.