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Protonation of the beta-lactam nitrogen is the trigger event in the catalytic action of class A beta-lactamases.

ABSTRACT: The pH dependence of the pK(a) values of all ionizable groups and of the electrostatic potential at grid points corresponding to catalytically important atoms in the active site of TEM-1 beta-lactamase has been calculated by a mean-field approach for reaction intermediates modeled on the basis of energy minimized x-ray crystallographic coordinates. By estimating electrostatic contributions to the free energy changes accompanying the conversion of the free enzyme into the acylenzyme reaction intermediate, we found that acid-catalyzed protonation of the beta-lactam nitrogen is energetically favored as the initiating event, followed by base-catalyzed nucleophilic attack on the carbonyl carbon of the beta-lactam group. N-protonation is catalyzed through a hydrogen-bonded cluster involving the 2-carboxylate group of the substrate, the side chains of S130 and K234, and a solvent molecule. Nucleophilic attack on the carbonyl carbon is carried out by the side chain of S70 with proton abstraction catalyzed by a water molecule hydrogen-bonded to the side chain of E166. Stabilization of ion pairs in the active site through interactions with distant clusters of charged residues in the enzyme was concluded to be an important driving force of the catalytic mechanism.

SUBMITTER: Atanasov BP

PROVIDER: S-EPMC16209 | biostudies-literature | 2000 Mar

REPOSITORIES: biostudies-literature

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Protonation of the beta-lactam nitrogen is the trigger event in the catalytic action of class A beta-lactamases.

Atanasov B P BP Mustafi D D Makinen M W MW

Proceedings of the National Academy of Sciences of the United States of America 20000301 7

The pH dependence of the pK(a) values of all ionizable groups and of the electrostatic potential at grid points corresponding to catalytically important atoms in the active site of TEM-1 beta-lactamase has been calculated by a mean-field approach for reaction intermediates modeled on the basis of energy minimized x-ray crystallographic coordinates. By estimating electrostatic contributions to the free energy changes accompanying the conversion of the free enzyme into the acylenzyme reaction inte ...[more]

PMID: 10716727

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Project description:OXA beta-lactamases are largely responsible for beta-lactam resistance in Acinetobacter spp. and Pseudomonas aeruginosa, two of the most difficult-to-treat nosocomial pathogens. In general, the beta-lactamase inhibitors used in clinical practice (clavulanic acid, sulbactam, and tazobactam) demonstrate poor activity against class D beta-lactamases. To overcome this challenge, we explored the abilities of beta-lactamase inhibitors of the C-2- and C-3-substituted penicillin and cephalosporin sulfone families against OXA-1, extended-spectrum (OXA-10, OXA-14, and OXA-17), and carbapenemase-type (OXA-24/40) class D beta-lactamases. Three C-2-substituted penicillin sulfone compounds (JDB/LN-1-255, JDB/LN-III-26, and JDB/ASR-II-292) showed low K(i) values for the OXA-1 beta-lactamase (0.70 +/- 0.14 --> 1.60 +/- 0.30 microM) and demonstrated significant K(i) improvements compared to the C-3-substituted cephalosporin sulfone (JDB/DVR-II-214), tazobactam, and clavulanic acid. The C-2-substituted penicillin sulfones JDB/ASR-II-292 and JDB/LN-1-255 also demonstrated low K(i)s for the OXA-10, -14, -17, and -24/40 beta-lactamases (0.20 +/- 0.04 --> 17 +/- 4 microM). Furthermore, JDB/LN-1-255 displayed stoichiometric inactivation of OXA-1 (the turnover number, i.e., the partitioning of the initial enzyme inhibitor complex between hydrolysis and enzyme inactivation [t(n)] = 0) and t(n)s ranging from 5 to 8 for the other OXA enzymes. Using mass spectroscopy to study the intermediates in the inactivation pathway, we determined that JDB/LN-1-255 inhibited OXA beta-lactamases by forming covalent adducts that do not fragment. On the basis of the substrate and inhibitor kinetics of OXA-1, we constructed a model showing that the C-3 carboxylate of JDB/LN-1-255 interacts with Ser115 and Thr213, the R-2 group at C-2 fits between the space created by the long B9 and B10 beta strands, and stabilizing hydrophobic interactions are formed between the pyridyl ring of JDB/LN-1-255 and Val116 and Leu161. By exploiting conserved structural and mechanistic features, JDB/LN-1-255 is a promising lead compound in the quest for effective inhibitors of OXA-type beta-lactamases.

Dataset Information

Protonation of the beta-lactam nitrogen is the trigger event in the catalytic action of class A beta-lactamases.

Publications

Protonation of the beta-lactam nitrogen is the trigger event in the catalytic action of class A beta-lactamases.

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