Some differences in turnover kinetics of penams and cephems catalyzed by classes A and C ?-lactamases

Abstract

The differences in the kinetic mechanism of catalysis of class A and class C ?-lactamases are shown by the manner by which these enzymes catalyze the hydrolyses of the penicillins and the cephalosporins. The hydrolysis of cephalosporins that are good substrates of the class C P99 ?-lactamase, where deacylation of an acyl-enzyme intermediate is rate-determining, has been shown to involve a free enzyme isoform (Adediran et al, 2021). We describe here how reconversion of this isoform to the native enzyme is accelerated by bases (e.g. imidazole) and salts (e. g. sodium chloride). The hydrolysis of penicillins by the P99 enzyme, where deacylation is also rate-determining, is not affected by imidazole and sodium chloride, a result that suggests that an enzyme isoform does not accumulate as an intermediate in turnover of this class of substrate. In support of these conclusions, solvent deuterium kinetic isotope effects on k_cat values were changed by the presence of imidazole for turnover of cephalothin by the P99 enzyme but unaffected for benzylpenicillin turnover. The hydrolyses of cephalosporins and penicillins by the class A TEM-2 ?-lactamase were not affected by imidazole and sodium chloride and thus also may not involve an accumulating free enzyme isoform. Solvent deuterium kinetic isotope effects and proton inventories on the class A PC1 and P99 ?-lactamase-catalyzed hydrolyses of benzylpenicillin at saturating concentrations showed the deacylation transition states of these two classes of enzymes to be different with respect to proton motion.