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

Abstract

The kinetic details of the irreversible inactivation of the E. coli RTEM .beta.-lactamase by clavulanic acid [from Streptomyces clavuligerus] were elucidated. Clavulanate is destroyed by the enzyme and simultaneously inhibits it by producing 2 catalytically inactive forms. One of these is transiently stable and decomposes to free enzyme (k = 3.8 .times. 10-3 s-1), while the other corresponds to an irreversibly inactivated form. The transient complex is formed from the Michaelis complex at a rate (k .apprx. 3 .times. 10-2 s-1) which is some 3-fold faster than the rate of formation of the irreversibly inactivated complex. The transient complex is, therefore, the principle enzyme form present after short time periods. In the presence of excess clavulanate, all the enzyme accumulates into the irreversibly inactivated form. The number of clavulanate turnovers that occur prior to complete enzyme inactivation is 115.