Biapenem Inactivation by B2 Metallo β-Lactamases: Energy Landscape of the Post-Hydrolysis Reactions

<div><h3>Background</h3><p>The first line of defense by bacteria against <em>β</em>-lactam antibiotics is the expression of β-lactamases, which cleave the amide bond of the β-lactam ring. In the reaction of biapenem inactivation by B2 metallo β-lactamases (MβLs), after the β-lactam ring is opened, the carboxyl group generated by the hydrolytic process and the hydroxyethyl group (common to all carbapenems) rotate around the C5–C6 bond, assuming a new position that allows a proton transfer from the hydroxyethyl group to C2, and a nucleophilic attack on C3 by the oxygen atom of the same side-chain. This process leads to the formation of a bicyclic compound, as originally observed in the X-ray structure of the metallo β-lactamase CphA in complex with product.</p> <h3>Methodology/Principal Findings</h3><p>QM/MM and metadynamics simulations of the post-hydrolysis steps in solution and in the enzyme reveal that while the rotation of the hydroxyethyl group can occur in solution or in the enzyme active site, formation of the bicyclic compound occurs primarily in solution, after which the final product binds back to the enzyme. The calculations also suggest that the rotation and cyclization steps can occur at a rate comparable to that observed experimentally for the enzymatic inactivation of biapenem only if the hydrolysis reaction leaves the N4 nitrogen of the β-lactam ring unprotonated.</p> <h3>Conclusions/Significance</h3><p>The calculations support the existence of a common mechanism (in which ionized N4 is the leaving group) for carbapenems hydrolysis in all MβLs, and suggest a possible revision of mechanisms for B2 MβLs in which the cleavage of the β-lactam ring is associated with or immediately followed by protonation of N4. The study also indicates that the bicyclic derivative of biapenem has significant affinity for B2 MβLs, and that it may be possible to obtain clinically effective inhibitors of these enzymes by modification of this lead compound.</p> </div

A tetrahedral intermediate in the aminolysis of benzylpenicillin

There is a non-linear dependent of the rate of aminolysis of benzylpenicillin upon hydroxide ion concentration which is interpreted in terms of formation of a tetrahedral intermediate; the rate of breakdown of the intermediate into reactants is ca. 109 s–

The effect of increasing the hydrophobicity of penicillin on its micelle-catalysed hydrolysis

Micelles of cetyltrimethylammonium bromide catalyse the alkaline hydrolysis of alkylpenicillins and benzylpenicillin methyl ester, The equilibrium constant for binding the alkylpenicillin to the micelle and the rate constants have been obtained. Binding increases with increasing alkyl chain length but shows a non-linear dependence upon the Hansch -value and the rate reaches a maximum value with heptylpenicillin. The free energy of transfer of a methylene group from water to the micelle shows a maximum value of 0.71 kcal mol–1. The negative charge of the carboxytate group of penicillin is not necessary tor catalysi

The aminolysis of penicillin derivatives. Rate constants for the formation and breakdown of the tetrahedral addition intermediate

There is a non-linear dependence of the rate of aminolysis of benzylpenicillin and 6--aminopenicillanic acid upon hydroxide ion concentration which is interpreted in terms of formation of a tetrahedral addition intermediate. At high concentrations of hydroxide ion the rate-limiting step is formation of the tetrahedral intermediate but at low concentrations it is the diffusion-controlled encounter of the intermediate and hydroxide ion. Rate constants for the formation of the intermediate and its breakdown to reactants are reported for a variety of amines. The dependence of these rate constants upon the pKa of the conjugate acid of the amine yield Brønsted values of ca. 0.3 and –0.6 for the formation and breakdown of the intermediate, respectively. There is thus quite a large dependence of the rate of expulsion of the amine from the intermediate upon the basicity of the amine despite the rate constants for this step being ca. 109–1010 s–1. Possible stereoelectronic control in the breakdown of the tetrahedral intermediate is discussed. There is no evidence for intramolecular general base catalysis in the formation of the tetrahedral intermediate from 6--aminopenicillanic aci