Chemical studies on the inactivation of Escherichia coli RTEM β-lactamase by clavulanic acid

Abstract

Incubation of clavulanic acid [from Streptomyces clavuligerus] with the .beta.-lactamase from E. coli RTEM leads to enzyme-catalyzed depletion of clavulanic acid, to transient inhibition and to irreversible inactivation of the enzyme. Both the transiently inhibited and the irreversibly inactivated species show a marked increase in the absorbance at 281 nm that is proportional to the decrease in enzyme activity. Hydroxylamine treatment of irreversibly inactivated enzyme restores about 1/3 of the catalytic activity, with a concomitant decrease in absorbance at 281 nm. Polyacrylamide isoelectric focusing of the irreversibly inactivated enzyme shows 3 bands of approximately equal intensity, different from native enzyme. Upon hydroxylamine treatment, 1 of the 3 bands disappears and now focuses identically with native enzyme. Apparently, the irreversible inactivation of enzyme by an excess of clavulanic acid generates 3 products, 1 of which can be reactivated by hydroxylamine.