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Delta 2- and delta 3-cephalosporins, penicillinate and 6-unsubstituted penems. Intrinsic reactivity and interaction with beta-lactamases and D-alanyl-D-alanine-cleaving serine peptidases.

ABSTRACT: The intrinsic reactivity of delta 2- and delta 3-deacetoxy-7-phenylacetamidocephalosporanates, penicillanate, unsubstituted, 2-methyl- and 2-phenyl-penems and other beta-lactam antibiotics has been expressed in terms of the second-order rate constant (M-1.s-1(OH-)) for the hydrolysis of the beta-lactam amide bond by OH- at 37 degrees C. The values thus obtained have been compared with the second-order rate constants (M-1.s-1(enzyme) for the opening of the same beta-lactam amide bond during interaction with the beta-lactamases of Streptomyces albus G and Actinomadura R39 and the D-alanyl-D-alanine-cleaving serine peptidases of Streptomyces R61 and Actinomadura R39. Depending on the cases, the accelerating effect due to enzyme action and expressed by the ratio M-1.s-1(enzyme)/M-1.s-1(OH) varies from less than 2 to more than 1 x 10(6). The primary parameter that governs enzyme action is the goodness of fit of the beta-lactam molecule to the enzyme cavity rather than its intrinsic reactivity. With the D-alanyl-D-alanine-cleaving serine peptidases, the three penems studied form intermediate complexes characterized by very short half lives of 14-100 s, values significantly lower than those exhibited by most beta-lactam compounds.

SUBMITTER: Frere JM

PROVIDER: S-EPMC1158213 | biostudies-other | 1982 Apr

REPOSITORIES: biostudies-other

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Project description:The biochemical properties of CMY-32, a class C enzyme possessing a single-amino acid substitution in the Omega loop (Gly214Glu), were compared to those of the parent enzyme, CMY-2, a widespread class C beta-lactamase. In parallel with our microbiological characterization, the Gly214Glu substitution in CMY-32 reduced catalytic efficiency (k(cat)/K(m)) by 50-70% against "good" substrates (i.e., cephalothin) while increasing k(cat)/K(m) against "poor" substrates (i.e., cefotaxime). Additionally, CMY-32 was more susceptible to inactivation by sulfone beta-lactamase inhibitors (i.e., sulbactam and tazobactam) than CMY-2. Timed electrospray ionization mass spectrometry (ESI-MS) analysis of the reaction of CMY-2 and CMY-32 with different substrates and inhibitors suggested that both beta-lactamases formed similar intermediates during catalysis and inactivation. We next showed that the carbapenems (imipenem, meropenem, and doripenem) form long-lived acyl-enzyme intermediates and present evidence that there is beta-lactamase-catalyzed elimination of the C(6) hydroxyethyl substituent. Furthermore, we discovered that the monobactam aztreonam and BAL29880, a new beta-lactamase inhibitor of the monobactam class, inactivate CMY-2 and CMY-32 by forming an acyl-enzyme intermediate that undergoes elimination of SO(3)(2-). Molecular modeling and dynamics simulations suggest that the Omega loop is more constrained in CMY-32 than CMY-2. Our model also proposes that Gln120 adopts a novel conformation in the active site while new interactions form between Glu214 and Tyr221, thus explaining the increased level of cefotaxime hydrolysis. When it is docked in the active site, we observe that BAL29880 exploits contacts with highly conserved residues Lys67 and Asn152 in CMY-2 and CMY-32. These findings highlight (i) the impact of single-amino acid substitutions on protein evolution in clinically important AmpC enzymes and (ii) the novel insights into the mechanisms by which carbapenems and monobactams interact with CMY-2 and CMY-32 beta-lactamases.

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Delta 2- and delta 3-cephalosporins, penicillinate and 6-unsubstituted penems. Intrinsic reactivity and interaction with beta-lactamases and D-alanyl-D-alanine-cleaving serine peptidases.

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