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Kinetics and mechanism of inhibition of a serine beta-lactamase by O-aryloxycarbonyl hydroxamates.

ABSTRACT: The class C serine beta-lactamase of Enterobacter cloacae P99 is irreversibly inhibited by O-aryloxycarbonyl hydroxamates. A series of these new inhibitors has been prepared to investigate the kinetics and mechanism of the inactivation reaction. A pH-rate profile for the reaction indicated that the reactive form of the inhibitor is neutral rather than anionic. The reaction rate is enhanced by electron-withdrawing aryloxy substituents and by hydrophobic substitution on both aryloxy and hydroxamate groups. Kinetics studies show that the rates of loss of the two possible leaving groups, aryloxide and hydroxamate, are essentially the same as the rate of enzyme inactivation. Nucleophilic trapping experiments prove, however, that the aryl oxide is the first to leave. It is likely, therefore, that the rate-determining step of inactivation is the initial acylation reaction, most likely of the active site serine, yielding a hydroxamoyl-enzyme intermediate. This then partitions between hydrolysis and aminolysis by Lys 315, the latter to form an inactive, cross-linked active site. A previously described crystal structure of the inactivated enzyme shows a carbamate cross-link of Ser 64 and Lys 315. Structure-activity studies of the reported compounds suggest that they do not react at the enzyme active site in the same way as normal substrates. In particular, it appears that the initial acylation by these compounds does not involve the oxyanion hole, an unprecedented departure from known and presumed reactivity. Molecular modeling suggests that an alternative oxyanion hole may have been recruited, consisting of the side chain functional groups of Tyr 150 and Lys 315. Such an alternative mode of reaction may lead to the design of novel inhibitors.

SUBMITTER: Pelto RB

PROVIDER: S-EPMC2669106 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Kinetics and mechanism of inhibition of a serine beta-lactamase by O-aryloxycarbonyl hydroxamates.

Pelto Ryan B RB Pratt R F RF

Biochemistry 20081023 46

The class C serine beta-lactamase of Enterobacter cloacae P99 is irreversibly inhibited by O-aryloxycarbonyl hydroxamates. A series of these new inhibitors has been prepared to investigate the kinetics and mechanism of the inactivation reaction. A pH-rate profile for the reaction indicated that the reactive form of the inhibitor is neutral rather than anionic. The reaction rate is enhanced by electron-withdrawing aryloxy substituents and by hydrophobic substitution on both aryloxy and hydroxamat ...[more]

PMID: 18942857

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Project description:β-Lactamases hydrolyze β-lactam antibiotics and are major determinants of antibiotic resistance in Gram-negative pathogens. Enmetazobactam (formerly AAI101) and tazobactam are penicillanic acid sulfone (PAS) β-lactamase inhibitors that differ by an additional methyl group on the triazole ring of enmetazobactam, rendering it zwitterionic. In this study, ultrahigh-resolution X-ray crystal structures and mass spectrometry revealed the mechanism of PAS inhibition of CTX-M-15, an extended-spectrum β-lactamase (ESBL) globally disseminated among Enterobacterales. CTX-M-15 crystals grown in the presence of enmetazobactam or tazobactam revealed loss of the Ser70 hydroxyl group and formation of a lysinoalanine cross-link between Lys73 and Ser70, two residues critical for catalysis. Moreover, the residue at position 70 undergoes epimerization, resulting in formation of a d-amino acid. Cocrystallization of enmetazobactam or tazobactam with CTX-M-15 with a Glu166Gln mutant revealed the same cross-link, indicating that this modification is not dependent on Glu166-catalyzed deacylation of the PAS-acylenzyme. A cocrystal structure of enmetazobactam with CTX-M-15 with a Lys73Ala mutation indicates that epimerization can occur without cross-link formation and positions the Ser70 Cβ closer to Lys73, likely facilitating formation of the Ser70-Lys73 cross-link. A crystal structure of a tazobactam-derived imine intermediate covalently linked to Ser70, obtained after 30 min of exposure of CTX-M-15 crystals to tazobactam, supports formation of an initial acylenzyme by PAS inhibitors on reaction with CTX-M-15. These data rationalize earlier results showing CTX-M-15 deactivation by PAS inhibitors to involve loss of protein mass, and they identify a distinct mechanism of β-lactamase inhibition by these agents. IMPORTANCE β-Lactams are the most prescribed antibiotic class for treating bacterial diseases, but their continued efficacy is threatened by bacterial strains producing β-lactamase enzymes that catalyze their inactivation. The CTX-M family of ESBLs are major contributors to β-lactam resistance in Enterobacterales, preventing effective treatment with most penicillins and cephalosporins. Combining β-lactams with β-lactamase inhibitors (BLIs) is a validated route to overcome such resistance. Here, we describe how exposure to enmetazobactam and tazobactam, BLIs based on a penicillanic acid sulfone (PAS) scaffold, leads to a protein modification in CTX-M-15, resulting in irremediable inactivation of this most commonly encountered member of the CTX-M family. High-resolution X-ray crystal structures showed that PAS exposure induces formation of a cross-link between Ser70 and Lys73, two residues critical to β-lactamase function. This previously undescribed mechanism of inhibition furthers our understanding of β-lactamase inhibition by classical PAS inhibitors and provides a basis for further, rational inhibitor development.

Project description:This paper reviews previously published data and presents new results to address the hypothesis that fluorinated aminophosphonates (FAPs), (RO)(2)P(O)C(CF(3))(2)NHS(O)(2)C(6)H(5), R=alkyl, inhibit serine esterases by scission of the P-C bond. Kinetics studies demonstrated that FAPs are progressive irreversible inhibitors of acetylcholinesterase (AChE, EC 3.1.1.7.), butyrylcholinesterase (BChE, EC 3.1.1.8.), carboxylesterase (CaE, EC 3.1.1.1.), and neuropathy target esterase (NTE, EC 3.1.1.5.), consistent with P-C bond breakage. Chemical reactivity experiments showed that diMe-FAP and diEt-FAP react with water to yield the corresponding dialkylphosphates and (CF(3))(2)CHNHS(O)(2)C(6)H(5), indicating lability of the P-C bond. X-ray crystallography of diEt-FAP revealed an elongated (and therefore weaker) P-C bond (1.8797 (13)A) compared to P-C bonds in dialkylphosphonates lacking alpha-CF(3) groups (1.805-1.822A). Semi-empirical and non-empirical molecular modeling of diEt-FAP and (EtO)(2)P(O)C(CH(3))(2)NHS(O)(2)C(6)H(5) (diEt-AP), which lacks CF(3) groups, indicated lengthening and destabilization of the P-C bond in diEt-FAP compared to diEt-AP. Active site peptide adducts formed by reacting diEt-FAP with BChE and diBu-FAP with NTE catalytic domain (NEST) were identified using peptide mass mapping with mass spectrometry (MS). Mass shifts (mean+/-SE, average mass) for peaks corresponding to active site peptides with diethylphosphoryl and monoethylphosphoryl adducts on BChE were 136.1+/-0.1 and 108.0+/-0.1Da, respectively. Corresponding mass shifts for dibutylphosphoryl and monobutylphosphoryl adducts on NEST were 191.8+/-0.2 and 135.5+/-0.1Da, respectively. Each of these values was statistically identical to the theoretical mass shift for each dialkylphosphoryl and monoalkylphosphoryl species. The MS results demonstrate that inhibition of BChE and NEST by FAPs yields dialkylphosphoryl and monoalkylphosphoryl adducts, consistent with phosphorylation via P-C bond cleavage and aging by net dealkylation. Taken together, predictions from enzyme kinetics, chemical reactivity, X-ray crystallography, and molecular modeling were confirmed by MS and support the hypothesis that FAPs inhibit serine esterases via scission of the P-C bond.

Dataset Information

Kinetics and mechanism of inhibition of a serine beta-lactamase by O-aryloxycarbonyl hydroxamates.

Publications

Kinetics and mechanism of inhibition of a serine beta-lactamase by O-aryloxycarbonyl hydroxamates.

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