Kinetic studies on inactivation of Citrobacter freundii cephalosporinase by sulbactam

The inactivation kinetics for inhibition by sulbactam (CP45,899) of Citrobacter freundii GN346 cephalosporinase were studied in detail and compared with those of type Ib penicillinase or TEM-2 beta-lactamase mediated by R plasmid RGN823. The rate constant for progressive inactivation of the cephalosporinase was significantly larger than that measured with the penicillinase. The number of sulbactam molecules required to cause complete inactivation of one cephalosporinase molecule (turnover number) was 80. The turnover number for the penicillinase was 5,200. The powerful inhibition by sulbactam of this cephalosporinase is similar to clavulanic acid inhibition of the penicillinase (turnover number, 115; reported by others). The affinity of sulbactam for the cephalosporinase, expressed as Ki, was 500 microM; this value was much higher than that for the penicillinase, which was estimated to be 0.5 microM. These results indicated that sulbactam is an effective progressive inactivator but a poor competitive inhibitor for the cephalosporinase. Our study also revealed that the cephalosporinase and sulbactam formed a long-lived inhibitor-enzyme complex which we termed the pseudo-irreversible complex. The half-life of the complex was 550 min at pH 7.0 and 30 degrees C.