M. Nukaga et al., INTERACTION OF OXYIMINO BETA-LACTAMS WITH A CLASS-C BETA-LACTAMASE AND A MUTANT WITH A SPECTRUM EXTENDED TO BETA-LACTAMS, Antimicrobial agents and chemotherapy, 38(6), 1994, pp. 1374-1377
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.