title: A New Covalent Inhibitor of Class C β-Lactamases Reveals Extended Active Site Specificity.
abstract: O-Aryloxycarbonyl hydroxamates have previously been shown to efficiently inactivate class C β-lactamases by cross-linking serine and lysine residues in the active site. A new analogue of these inhibitors, D-(R)-O-(phenoxycarbonyl)-N-[(4-amino-4-carboxy-1-butyl)oxycarbonyl]hydroxylamine, designed to inactivate certain low-molecular mass dd-peptidases, has now been synthesized. Although the new molecule was found to be only a poor inactivator of the latter enzymes, it proved, unexpectedly, to be a very effective inactivator (ki = 3.5 × 10(4) M(-1) s(-1)) of class C β-lactamases, more so than the original lead compound, O-phenoxycarbonyl-N-(benzyloxycarbonyl)hydroxylamine. Furthermore, the mechanism of inactivation is different. Mass spectrometry demonstrated that β-lactamase inactivation by the new molecule involved formation of an O-alkoxycarbonylhydroxamate with the nucleophilic active site serine residue. This acyl-enzyme did not cyclize to cross-link the active site as did that from the lead compound. Model building suggested that the rapid enzyme acylation by the new molecule may occur because of favorable interaction between the polar terminus of its side chain and elements of the Ω loop that abuts the active site, Arg 204 in particular. This interaction should be considered in the design of new covalent β-lactamase inhibitors. The initially formed acyl-enzyme partitions (ratio of ∼ 1) between hydrolysis, which regenerates the active enzyme, and formation of an inert second acyl-enzyme. Structural modeling suggests that the latter intermediate arises from conformational movement of the acyl group away from the reaction center, probably enforced by the inflexibility of the acyl group. The new molecule is thus a mechanism-based inhibitor in which an inert complex is formed by noncovalent rearrangement. Phosphyl analogues of the new molecule were efficient inactivators of neither dd-peptidases nor β-lactamases.
authors.affiliation: Department of Chemistry, Wesleyan University , Middletown, Connecticut 06459, United States.
authors.forename: Ronak
authors.initials: R
authors.lastname: Tilvawala
authors.affiliation: Department of Chemistry, University of Texas at Austin , Austin, Texas 78712-1224, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Cammarata
authors.affiliation: Department of Chemistry, Wesleyan University , Middletown, Connecticut 06459, United States.
authors.forename: S A
authors.initials: SA
authors.lastname: Adediran
authors.affiliation: Department of Chemistry, University of Texas at Austin , Austin, Texas 78712-1224, United States.
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Brodbelt
authors.affiliation: Department of Chemistry, Wesleyan University , Middletown, Connecticut 06459, United States.
authors.forename: R F
authors.initials: RF
authors.lastname: Pratt
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D013058:Mass Spectrometry; D065093:beta-Lactamase Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases