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 g lutamic acid at position 219 in the enzyme by lysine was previously sh own to broaden its substrate spectrum to oxyimino beta-lactams (K. Tsu kamoto, R. Ohno, and T. Sawai, J. Bacteriol. 172:4348-4351, 1990). To clarify this spectrum extension from the kinetic point of view, the in teractions of cefuroxime, ceftazidime, and aztreonam with the wild typ e and mutant enzymes were analyzed. In addition to aztreonam, known as a progressive inhibitor of class C beta-lactamases, cefuroxime and ce ftazidime were found to act as progressive inhibitors of the wild-type enzyme. On the other hand, only aztreonam showed weak progressive inh ibition of the mutant enzyme. On the basis of kinetic parameters, a mi nimum scheme for interaction of the oxyimino beta-lactams with the wil d-type enzyme was proposed, and the rate-limiting step of the hydrolys is of unfavorable substrates was indicated to be conversion of the sta ble acyl-enzyme intermediate to the unstable intermediate. In aztreona m hydrolysis by the mutant enzyme, the reaction rate at the rate-limit ing step was 2,000 times that of the wild-type enzyme. These results i ndicate that the mutation at position 219 disturbs the stabilization o f the stable intermediate.