Interaction of oxyimino beta-lactams with a class C beta-lactamase and a mutant with a spectrum extended to beta-lactams.
The class C beta-lactamase of Citrobacter freundii GN346 is a typical cephalosporinase comprising 361 amino acids, and substitution of the glutamic acid at position 219 in the enzyme by lysine was previously shown to broaden its substrate spectrum to oxyimino beta-lactams (K. Tsukamoto, R. Ohno, and T. Sawai, J. Bacteriol. 172:4348-4351, 1990). To clarify this spectrum extension from the kinetic point of view, the interactions of cefuroxime, ceftazidime, and aztreonam with the wild-type and mutant enzymes were analyzed. In addition to aztreonam, known as a progressive inhibitor of class C beta-lactamases, cefuroxime and ceftazidime were found to act as progressive inhibitors of the wild-type enzyme. On the other hand, only aztreonam showed weak progressive inhibition of the mutant enzyme. On the basis of kinetic parameters, a minimum scheme for interaction of the oxyimino beta-lactams with the wild-type enzyme was proposed, and the rate-limiting step of the hydrolysis of unfavorable substrates was indicated to be conversion of the stable acyl-enzyme intermediate to the unstable intermediate. In aztreonam hydrolysis by the mutant enzyme, the reaction rate at the rate-limiting step was 2,000 times that of the wild-type enzyme. These results indicate that the mutation at position 219 disturbs the stabilization of the stable intermediate.
Nukaga
M
M
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Tsukamoto
K
K
Yamaguchi
H
H
Sawai
T
T
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001398:Aztreonam / Q000494:pharmacology; D007700:Kinetics; D009154:Mutation; D065093:beta-Lactamase Inhibitors
D000900:Anti-Bacterial Agents; D065093:beta-Lactamase Inhibitors; D001398:Aztreonam