Negative impact of carbapenem methylation on the reactivity of β-lactams for cysteine acylation revealed by quantum calculations and kinetic analyses.

The Enterococcus faecium L,D-transpeptidase (Ldt(fm)) mediates resistance to most beta-lactam antibiotics in this bacterium by replacing classical peptidoglycan polymerases. The catalytic Cys of Ldt(fm) is rapidly acylated by beta-lactams belonging to the carbapenem class but not by penams or cephems. We previously reported quantum calculations and kinetic analyses for Ldt(fm) and showed that the inactivation profile is not determined by differences in drug binding (K-D [equilibrium dissociation constant] values in the 50 to 80 mM range). In this study, we analyzed the reaction of a Cys sulfhydryl with various beta-lactams in the absence of the enzyme environment in order to compare the intrinsic reactivity of drugs belonging to the penam, cephem, and carbapenem classes. For this purpose, we synthesized cyclic Cys-Asn (cCys-Asn) to generate a soluble molecule with a sulfhydryl closely mimicking a cysteine in a polypeptide chain, thereby avoiding free reactive amino and carboxyl groups. Computational studies identified a thermodynamically favored pathway involving a concerted rupture of the beta-lactam amide bond and formation of an amine anion. Energy barriers indicated that the drug reactivity was the highest for non-methylated carbapenems, intermediate for methylated carbapenems and cephems, and the lowest for penams. Electron-withdrawing groups were key reactivity determinants by enabling delocalization of the negative charge of the amine anion. Acylation rates of cCys-Asn determined by spectrophotometry revealed the same order in the reactivity of beta-lactams. We concluded that the rate of Ldt(fm) acylation is largely determined by the beta-lactam reactivity with one exception, as the enzyme catalytic pocket fully compensated for the detrimental effect of carbapenem methylation.