pH Dependence of and kinetic solvent isotope effects on the methanolysis and hydrolysis of .beta.-lactams catalyzed by class C .beta.-lactamase

Page, Michael I.; Vilanova, Bartolomé; Layland, Nicola J.

doi:10.1021/ja00154a009

Cited by 34 publications

(49 citation statements)

References 5 publications

(5 reference statements)

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“…Similar pKa values have been reported for reactions of the P99 β-lactamase (kcat/Km) with substrates, e.g. 5.85 ± 0.09, 5.92 ± 0.16, 6.21 ± 0.11, 5.89 ± 0.07 and 6.29 ± 0.04 (24,25). The important consequence of the pH profile of Figure 3, which is presumably very similar to those for 3 – 8 because of their similar pK a2 values (see above), is that the maximal activity of these inhibitors is expressed at around pH 6.2 and thus the data of Table1, taken at pH 7.5, does not reflect their absolute potency [e.g.…”

Section: Resultssupporting

confidence: 83%

“…The value of pK a2 strongly suggests that the second dissociation in the pH-rate profile of 1 (K a2 ) represents dissociation of the inhibitor rather than that of the enzyme, as represented in Scheme 5a. Although normal substrates also display a second dissociation associated with loss of enzyme activity at high pH, pKa values for this dissociation are typically around 9 (25,26). It seems likely, therefore, that the active inhibitor in the present case is the neutral compound rather than the anion.…”

Section: Resultsmentioning

confidence: 99%

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Kinetics and Mechanism of Inhibition of a Serine β-Lactamase by O-Aryloxycarbonyl Hydroxamates

Pelto

Pratt

2008

Biochemistry

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The class C serine β-lactamase of Enterobacter cloacae P99 is irreversibly inhibited by Oaryloxycarbonyl 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 aryloxide 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.For decades now, β-lactams have been one of our most effective weapons against bacterial infections (1). These drugs, although still the first line of attack in many clinical situations, have been compromised to a considerable degree by bacterial resistance to them (2). Among various sources of resistance that have arisen in bacteria, the most generally troublesome is the production of β-lactamases. These enzymes very effectively catalyze the hydrolysis and thus destruction of β-lactams before they can reach their cellular targets (3).The threat posed by β-lactamases to the efficacy of β-lactam antibiotics has been tackled by pharmaceutical companies in several ways. One approach that has been quite successful to date is that of including a β-lactamase inhibitor with a β-lactam antibiotic in combination therapies. For many years now, such combinations, using the now-classical β-lactamase inhibitors clavulanic acid, sulbactam and tazobactam, have been used to advantage (4). Since these inhibitors are themselves β-lactams, however, it is perhaps not surprising to find that certain β-lactamase mutants are capable of hydrolyzing them quite effectively. Such mutants have †

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Section: Resultssupporting

confidence: 83%

Section: Resultsmentioning

confidence: 99%

Kinetics and Mechanism of Inhibition of a Serine β-Lactamase by O-Aryloxycarbonyl Hydroxamates

Pelto

Pratt

2008

Biochemistry

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“…6 (17). It is assumed that this pK a corresponds to that of the general base catalyst required for proton removal from serine 64 and used in the formation of the tetrahedral intermediate leading to acylation (10,17,18). Site-directed mutagenesis of Tyr-150 has not led to firm conclusions although it appears to be essential for hydrolysis of the best substrates (19).…”

Section: Resultsmentioning

confidence: 99%

“…The dependence of the rate constant, k cat /K m , for the P99 class C ␤-lactamase catalysed hydrolysis of benzylpenicillin on pH (Fig. 3) indicates that there are two ionisations which control hydrolytic catalytic activity corresponding to groups of pK a ca 6.1 and 10.1, representing the two protonic forms of the enzyme, EH 2 and EH, respectively (17). The catalytically important form of the enzyme for hydrolysis is thus EH.…”

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confidence: 99%

The Relative Catalytic Efficiency of β-Lactamase Catalyzed Acyl and Phosphyl Transfer

Slater

Laws

Page

2001

Bioorganic Chemistry

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“…The pK a of the ionising group acting as a general base obtained from pHrate profiles is 6.3 but the unusual shift in this pK a in D 2 O and the inverse kinetic solvent isotope effect on k cat =K m are indicative of a system with an abnormally high fractionation factor for the protonic state undergoing dissociation. 9 In common with serine proteases, the serine b-lactamases possess an oxyanion pocket which donates two hydrogen bonds to the b-lactam There is an obvious interest in finding new inhibitors of the b-lactamases and the sulfonation of serine enzymes offers an interesting but largely unexplored strategy for inhibition as an alternative to the traditional mechanism-based acylation process. In addition to their normal acyl substrates, many serine enzymes are known to react with other electrophilic centres such as phosphonyl derivatives.…”

Section: Introductionmentioning

confidence: 99%

Novel mechanism of inhibiting β-Lactamases by sulfonylation using β-Sultams

Page

Hinchliffe

Wood

et al. 2003

Bioorganic & Medicinal Chemistry Letters

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pH Dependence of and kinetic solvent isotope effects on the methanolysis and hydrolysis of .beta.-lactams catalyzed by class C .beta.-lactamase

Cited by 34 publications

References 5 publications

Kinetics and Mechanism of Inhibition of a Serine β-Lactamase by O-Aryloxycarbonyl Hydroxamates

Kinetics and Mechanism of Inhibition of a Serine β-Lactamase by O-Aryloxycarbonyl Hydroxamates

The Relative Catalytic Efficiency of β-Lactamase Catalyzed Acyl and Phosphyl Transfer

Novel mechanism of inhibiting β-Lactamases by sulfonylation using β-Sultams

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