β-Secondary and Solvent Deuterium Kinetic Isotope Effects on β-Lactamase Catalysis

Abstract

Beta-Secondary and solvent deuterium kinetic isotope effects have been determined for the steady-state kinetic parameters V/K and V for turnover of a depsipeptide substrate, m-[[(phenylacetyl)glycyl]-oxy]benzoic acid, and of a beta-lactam substrate, penicillanic acid, by three typical class A beta-lactamases and a class C beta-lactamase. The isotope effects on alkaline hydrolysis of these substrates have been used as a frame of reference. The effect of the transition state conformation of the substrates in determining the beta-secondary isotope effects has been explicitly considered. The inverse beta-secondary isotope effects on both V/K and V for the class A enzymes with both substrates indicate transition states where the carbonyl group of the scissile bond has become tetrahedral and therefore reflect typical acyl-transfer transition states. The solvent isotope effects indicate that enzyme deacylation (as reflected in V for the Staphylococcus aureus PC1 beta-lactamase) may be a classical general-base-catalyzed hydrolysis but that there is little proton motion in the enzyme acylation transition state (as revealed by V/K) for the TEM beta-lactamase and Bacillus cereus beta-lactamase I. These results provide kinetic support for the conjecture made on structural grounds that class A beta-lactamases employ an asymmetric double-displacement mechanism. The isotope effects on V/K for the class C beta-lactamase of Enterobacter cloacae P99 suggest an acyl-transfer transition state for the penicillin, although, as for the class A enzymes, without significant proton motion. On the other hand, the V/K transition state for depsipeptide does not seem to involve covalent chemistry. Suggestive of this conclusion are the measured beta-secondary isotope effect of 1,002 +/- 0.012 and the inverse solvent isotope effect. These results provide an example of a significant difference between the kinetics of turnover of a beta-lactam and a depsipeptide by a beta-lactamase. The V transition state for both substrates with the P99 beta-lactamase probably involves acyl-transfer (deacylation) where the conformation of the acyl-enzyme is closely restricted. The conformations of acyl-enzymes of the PC1 and P99 beta-lactamases correlate to the (different) dispositions of general base catalysts at their active sites.

Kinetic Characterization of Hydrolysis of Nitrocefin, Cefoxitin, and Meropenem by β-Lactamase fromMycobacterium tuberculosis

“…Fits of the data points yielded equivalent, small solvent kinetic isotope effects of 1.4 ± 0.1 on V and V/K (Table 1). The small effects on nitrocefin hydrolysis by BlaC are similar to those observed previously for penicillins and cephalosporins by Hardy & Kirsch (1.53 and 1.09, respectively) and Adediran & Pratt (1.04 and 1.39, respectively) (22, 30). Since k cat / K m only reports on steps between binding of nitrocefin to the free enzyme, and the formation of the covalent acyl-enzyme, the steady-state results for nitrocefin hydrolysis revealing equivalent, small solvent KIEs on k cat / K m and k cat supports acylation as the rate-limiting step for this substrate.…”

“…Solvent kinetic isotope effects are powerful probes of proton transfer events in enzyme-catalyzed reactions and have been used to probe transition state structures for both acylation and deacylation reactions of mechanism-related proteases (25–28). This extends to the analysis of SKIEs on β -lactamases, including important contributions on penicillanic acid sulfone hydrolysis by Brenner & Knowles (29), β -lactam hydrolysis by the Bacillus cereus TEM β -lactamase by Hardy & Kirsch (22) and β -lactam hydrolysis by several related class A and C β -lactamases by Adediran & Pratt (30). We chose three substrates for this analysis with BlaC, which span nearly five orders of catalytic turnover rates.…”

Ligand-Induced Proton Transfer and Low-Barrier Hydrogen Bond Revealed by X-ray Crystallography

“…3D). Previous kinetic isotope effect (KIE) studies and QM/MM calculations suggest that the formation of the tetrahedral transition state is the rate-limiting step of the acylation reaction during Class A β-lactamase catalysis 26,36 . More specifically, general base catalysis is required to initiate the formation of this tetrahedral TS, whereas general acid catalysis is involved in the collapse of the TS.…”

Reclaiming the Efficacy of β-Lactam–β-Lactamase Inhibitor Combinations: Avibactam Restores the Susceptibility of CMY-2-Producing Escherichia coli to Ceftazidime

“…The C-7 carbonyl of avibactam was positioned within the oxyanion hole formed by residues Ser64 (2.9 Å) and Ser318 (3.0 Å). We recognize that the general base involved in the acylation of avibactam is debated for class C ␤-lactamases (32); Tyr150 and Lys67 are hypothesized to be involved in the deprotonation of Ser64 (33)(34)(35)(36)(37)(38)(39)(40). The molecular representation generated here revealed that both Tyr150 (3.0 Å) and Lys67 (2.8 Å) can form hydrogenbonding interactions with the hydroxyl side chain of the nucleophilic residue Ser64, suggesting that either residue may be involved in the acylation mechanism of avibactam.…”