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

The Ldtfm L,D-transpeptidase mediates resistance to most β-lactam antibiotics in Enterococcus faecium by replacing classical peptidoglycan polymerases. The catalytic Cys of Ldtfm is rapidly acylated by β-lactams belonging to the carbapenem class but not by penams and cephems. We previously reported quantum calculations and kinetic analyses for Ldtfm and showed that the inactivation profile is not determined by differences in drug binding ( K D values in the 50-80 mM range). Here, we analyze the reaction of a Cys sulfhydryl with various β-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 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 β-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 β-lactams. We concluded that the rate of Ldtfm acylation is largely determined by the β-lactam reactivity with one exception as the enzyme catalytic pocket fully compensated for the detrimental effect of carbapenem methylation.