MECHANISM OF TURNOVER OF IMIPENEM BY THE TEM BETA-LACTAMASE REVISITED

The mechanism of turnover of imipenem, a clinically important carbapenem antibiotic, by the TEM-1 beta-lactamase has been investigated. The details of the turnover process appear to be more complicated than previously believed. The details of the mechanism of turnover have been explored by the study of energy-minimized structures for the two acyl-enzyme intermediates, for the two enzyme-product complexes, as well as kinetic experiments. Evidence is presented that the unusual stability of the enzyme-product complexes and a subtle protein conformational change in the course of turnover may play roles in the enzymic hydrolysis of imipenem. Furthermore, the rate of deacylation is the slow step in turnover. The slow rate of turnover has been explained by the function of the C-6 alpha hydroxyethyl group, which binds in a small pocket in the active site. The hydroxyl of the hydroxyethyl moiety makes a strong hydrogen bond to the side chain of Asn-132, whereby the methyl group of this moiety displaces the hydrolytic water molecule (i.e., Wat-712) by 2-3 Angstrom from its optimal position in the active site. This structural factor has been offered as the basis for turnover of imipenem being as much as 10(3)- to 10(4)-fold slower than with the better substrates for class A beta-lactamases.