Cefoxitin, N-formimidoyl thienamycin, clavulanic acid, and penicillanic acid sulfone as suicide inhibitors for different types of beta-lactamases produced by gram-negative bacteria

Abstract

Cefoxitin, N-formimidoyl thienamycin (MK0787), clavulanic acid, and penicillanic acid sulfone (CP45,899) were studied to determine their potency as suicide inhibitors of three very different kinds of beta-lactamases produced by Gram-negative bacteria: type Ib penicillinase (TEM-2 type), a typical cephalosporinase (the enzyme of Proteus morganii), and a cephalosporinase with broad substrate specificity (the enzyme of Proteus vulgaris). All these beta-lactams were confirmed to be quite stable to the three beta-lactamases. The absolute values of the turnover numbers (kcat) for these enzyme-catalyzed hydrolyses were determined, the values ranged from 0.25 minute-1 to 660 minute-1. All the beta-lactams studied, except cefoxitin, acted as suicide inhibitors of the typical cephalosporinase. Although cefoxitin did not exhibit such an effect, it was a powerful competitive inhibitor of this enzyme. Although the four beta-lactams acted as suicide inhibitors of the P. vulgaris cephalosporinase, the inactivated enzyme partially regained its activity after removing the effect of the free inhibitor. Type Ib penicillinase was also inactivated by the four beta-lactams, and the activity of the inactivated enzyme, except in the case of cefoxitin, was partially restored. The rate constants for enzyme inactivation or reactivation were calculated and are presented. The information obtained from this study suggests that the catalytic center of the P. vulgaris cephalosporinase is different not only from that of the penicillinase-type but also from that of the cephalosporinase-type beta-lactamases.