Investigations on beta-lactamase stability of recently developed beta-lactam compounds: study of enzyme kinetics.

The plasmid-mediated TEM-1 enzyme (E. coli K12 R6K) and chromosomally mediated enzymes from Enterobacter cloacae (IEP 8.5) and Citrobacter freundii (IEP 9.5) were highly purified. Enzyme kinetics were studied with various therapeutic compounds as substrates and lamoxactam, azthreonam, and N-formimidoyl thienamycin as inhibitors. Lamoxactam and azthreonam failed to inhibit the TEM-1 enzyme, whereas N-formimidoyl thienamycin was a competitive inhibitor. KI was 5 mumol/l--thus corresponding to KM--and independent of the substrate. With respect to chromosomally mediated enzymes, there was a time-dependent inhibition of beta-lactam hydrolysis. KI ranged from 0.06 to 0.2 mumol/l in dependence of the inhibitor. Enzyme kinetics suggested a non-competitive type of inhibition thus indicating that binding of these compounds to both chromosomally mediated enzymes must be followed by a further reaction step. It is evident that the stability against beta-lactamases of recently developed compounds is based on different mechanisms--characteristic for the enzyme being considered.