Negative Impact of Carbapenem Methylation on the Reactivity of β-Lactams for Cysteine Acylation as Revealed by Quantum Calculations and Kinetic Analyses

Abstract

The Enterococcus faecium L,D-transpeptidase (Ldt fm ) mediates resistance to most ␤-lactam antibiotics in this bacterium by replacing classical peptidoglycan polymerases. The catalytic Cys of Ldt fm is rapidly acylated by ␤-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 ␤-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 ␤-lactam amide bond and formation of an amine anion. Energy barriers indicated that the drug reactivity was the highest for nonmethylated 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 ␤-lactams. We concluded that the rate of Ldt fm acylation is largely determined by the ␤-lactam reactivity with one exception, as the enzyme catalytic pocket fully compensated for the detrimental effect of carbapenem methylation.

Synthesis of Carbapenems Containing Peptidoglycan Mimetics and Inhibition of the Cross‐Linking Activity of a Transpeptidase of l,d Specificity

“… Two‐step acylation of l,d ‐transpeptidase Ldt fm by carbapenems. The evidence for the formation of the amine anion in the first step of the acylation reaction is supported by previous studies [8, 43, 44] …”

“…Inactivation of l,d ‐transpeptidases was previously shown to be a two‐step reaction [8] starting with nucleophilic attack of the β‐lactam carbonyl by the sulfur of the catalytic cysteine (Scheme 5). This first step was proposed to lead to reversible formation of an amine anion [41] .…”

“…The efficacy of Ldt fm inactivation by peptidomimetic carbapenems was evaluated by determining the k 1 and k 2 kinetic constants for the formation of the amine anion and the acyl enzyme (Scheme 5), respectively [8, 17, 41, 44] . These kinetic constants were determined by stopped‐flow fluorimetry as the intrinsic fluorescence of the Ldt fm tryptophan residues varies for the free enzyme, the amine anion, and the acyl enzyme [41] .…”

“… [7] Optimization relied on introduction of various substituents at the C2 position and of a methyl at position C1. The latter modification increases the stability of the carbapenems with respect to hydrolysis by a host enzyme, albeit at the expense of a reduced reactivity of the drugs [8] . Carbapenems have been shown to be active against Mycobacterium tuberculosis in vitro [9, 10] and in vivo [11] and are part of the recommended treatment of pulmonary infections due to Mycobacterium abscessus in cystic fibrosis patients [12, 13] .…”

“…The latterm odification increases the stability of the carbapenems with respectt oh ydrolysis by ah ost enzyme, albeit at the expense of ar educed reactivity of the drugs. [8] Carbapenems have been shown to be active against Mycobacterium tuberculosis in vitro [9,10] and in vivo [11] and are part of the recommended treatment of pulmonary infections duet oMycobacterium abscessus in cystic fibrosis patients. [12,13] The antibacterial activity of carbapenems against M. tuberculosis and M. abscessus involves inactivation of multiple targets including PBPs and Ldts.…”