Kinetic and inhibition studies of Bacillus cereus beta-lactamase using a spectrophotometric method.

The use of a spectrophotometric method is reported for the characterization of a beta-lactamase (EC 3.5.2.6) from Bacillus cereus. Absorption coefficients of the mercaptides of various penicillins were determined with this method. The enzyme was kinetically characterized using penicillins. Inhibition studies with Bacillus cereus beta-lactamase and various penicillins showed a substrate type of inhibition, indicating an additional binding site for substrates without catalytic activity. The dissociation constant of this binding site was determined and the influence of this binding site upon the catalytic activity is discussed. Studies with beta-lactamase-stable penicillins as inhibitors and various penicillins showed different types of inhibition, which indicated the presence of an additional catalytically inactive binding site. Experiments with clavulanic acid, a beta-lactamase inhibitor without remarkable intrinsic antibacterial activity, showed a mixed type of inhibition. Based on the hypothesis for the existence of more than one substrate binding site on the enzyme, clavulanic acid was found to be bonded to both the catalytic active and the catalytic inactive binding site.