beta-Lactam antibiotics are generally thought to inhibit their target enzymes, the bacterial cell wall-synthesizing DD-peptidases, because of their resemblance to D-alanyl-D-alanine peptides. Although a favorable conformation of the latter does structurally resemble the beta-lactams with respect to backbone conformation, a significant difference is the presence of a D-methyl substituent on the penultimate alanine residue of the cell wall peptide. A classical beta-lactam antibiotic has a hydrogen in the corresponding position. In the process of evolution of a beta-lactamase from a DD-peptidase, it seems likely that this D-methyl group would be selected against, to ensure that the former enzyme would hydrolyze beta-lactams rather than peptides. In this paper, the effect of the penultimate D-alanine residue (as opposed to a glycine residue) has been examined in peptide substrates of a present-day DD-peptidase and a beta-lactamase. The peptides N-(phenylacetyl)-D-alanyl-D-phenylalanine and N-(phenylacetyl)glycyl-D-phenylalanine were used as a test pair against the DD-peptidase of Streptomyces R61 and the structurally very similar class C beta-lactamase of Enterobacter cloacae P99. The kinetics of turnover of both of these substrates were determined for both enzymes. To quantify the partitioning of the acyl-enzyme intermediate, the aminolysis by D-phenylalanine of a cognate pair of depsipeptides was also studied. Thus, free energy-reaction coordinate diagrams were constructed for turnover of both peptides by both enzymes. Comparison of these profiles showed that the D-methyl group is preferred over hydrogen by the DD-peptidase at all stages of catalysis (acyl-enzyme and acylation and deacylation transition states), whereas the beta-lactamase selects against the D-methyl group only at the peptide acylation transition state. A process of evolution by uniform dissociation of the methyl group by the beta-lactamase has apparently occurred. These results were explored structurally by computational models of the acylation tetrahedral intermediates. A methyl group pocket on the DD-peptidase, less favorable on the beta-lactamase, was identified. The interaction of the leaving group, the terminal D-alanine residue, with the two enzymes was interesting, since it seemed that different positively charged active site residues were directly associated with the carboxylate, Lys 315 in the beta-lactamase and Arg 285 (rather than His 298) in the case of the DD-peptidase. The problems posed by larger substituents on the penultimate residue of the peptide, and in particular by the heterocyclic substituent present in a bicyclic beta-lactam, were analyzed. Qualitative and quantitative analysis of the models support the proposed importance of the penultimate D-alanine in beta-lactamase evolution.

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 series of acyclic substrates by the DD-peptidase of Streptomyces R61 and the class C beta-lactamase of Enterobacter cloacae P99. Although these enzymes are evolutionarily related and have very similar tertiary and active site structure, they are functionally very different-the former efficiently catalyzes the hydrolysis of beta-lactams but not acyclic peptides while vice versa applies to the latter. The measured kinetic isotope effects reveal both similarities and differences in the steady-state transition states for turnover of the various substrates by these enzymes. In most cases, inverse beta-secondary isotope effects were observed, reflecting typical acyl-transfer transition states. With one substrate, however, m-[[(phenylacetyl)glycyl]oxy]benzoic acid, isotope effects on V/K of very close to unity were obtained for both enzymes. These were interpreted in terms of acylation transition state conformations where the extent of beta-CH hyperconjugation was similar to that in the free substrate. Differences in deacylation transition states (V) between the two enzymes with this substrate were interpreted in terms of different acyl-enzyme conformations. Solvent deuterium kinetic isotope effects on V/K were uniformly small, some even inverse, for both enzymes and with all substrates tested. At face value, this suggests the counterintuitive conclusion that little proton transfer occurs in acylation transition states in all of these instances. Closer analysis, however, suggests that for ester and amide (and probably beta-lactam) substrates, this result probably arises from an increase in proton fractionation factors on substrate binding being offset by their decrease in the acylation transition state. The former event derives from proton rearrangement on substrate binding and the latter, presumably, from general acid/base catalysis. This result may be general to all beta-lactam-recognizing enzymes. The solvent isotope effects also suggest that, at least for the P99 beta-lactamase, the acylation transition state of a thioester substrate does not involve proton transfer. This can be interpreted in terms of the rate-determining breakdown of a tetrahedral intermediate where no protonation of the leaving thiolate is required. Deacylation transition states of both enzymes appear to involve significant proton transfer, presumably arising from general acid/base catalysis.

The kinetics of the inactivation of Bacillus cereus beta-lactamase I by 6 beta-bromopenicillanic acid are described. Loss of beta-lactamase activity is accompanied by a decrease in protein fluorescence, by the appearance of a protein-bound chromophore at 326 nm, and by loss of tritium from 6 alpha-[3H]-6 beta-bromopenicillanic acid. It is shown that all of the above changes probably have the same rate-determining step. The inactivation reaction is competitively inhibited by cephalosporin C, a competitive inhibitor of this enzyme, and by covalently bound clavulanic acid, suggesting that 6 beta-bromopenicillanic acid reacts directly with the beta-lactamase active site. It is proposed that this inhibitor reacts initially as a normal substrate and that the rate-determining step of the inactivation is acylation of the enzyme. A rapid irreversible inactivation reaction rather than normal hydrolysis of the acyl-enzyme then follows acylation; 6 beta-bromopenicillanic acid is thus a suicide substrate.

Aryl malonamates are demonstrated to be novel substrates of a broad range of beta-lactam-recognizing enzymes. These compounds are isomers of the aryl phenaceturates, which are well-known substrates of these enzymes, but the new compounds contain a retro-amide side chain. Several lines of evidence, including comparisons of steady-state kinetic parameters between enzymes and a detailed investigation of the methanolysis kinetics, solvent deuterium isotope effects, and pH-rate profile for turnover of a retro substrate by the Enterobacter cloacae P99 beta-lactamase, suggested that the new substrates are likely to be hydrolyzed by the same chemical mechanisms as "normal" substrates. Molecular modeling indicated that the retro-amide group fits snugly into the active site of the P99 beta-lactamase by hydrogen bonding to the conserved lysine-67 residue. The retro-amide side chain may represent a lead to novel mechanism-based and transition state analogue inhibitors.