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The inhibition of staphylococcal beta-lactamase by clavulanic acid.

ABSTRACT: Clavulanic acid inhibited both the extracellular and cell-extract beta-lactamases of the four Staphylococcus aureus strains tested. The inhibition of S. aureus Russell cell-extract enzyme appeared to be active-site-directed and proceeded in a first-order fashion consistent with the formation of a covalent intermediate. Inhibited enzyme free of excess clavulanic acid was shown to regenerate enzyme activity slowly at pH 7.0, but the rate of reactivation increased at acid pH. When the enzyme was incubated with excess clavulanic acid complete inhibition was rapidly obtained, during further incubation clavulanic acid was shown to disappear slowly and complete loss of clavulanic acid from the reaction mixture coincided with the onset of the return of enzyme activity. A reactive enamine resulting from enzymic hydrolysis of the beta-lactam ring of clavulanic acid has been proposed as a possible intermediate in the inhibitory mechanism.

SUBMITTER: Reading C

PROVIDER: S-EPMC1186596 | biostudies-other | 1979 Apr

REPOSITORIES: biostudies-other

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Project description:The beta-lactamase from Klebsiella pneumoniae E70 behaved in a similar fashion to the TEM-2 plasmid mediated enzyme on reaction with clavulanic acid. Both enzymes produced two types of enzyme-clavulanate complex, a transiently stable species (t((1/2))=4min at pH7.3 and 37 degrees C) and irreversibly inhibited enzyme. In the initial rapid reaction (2.5min) the enzymes partitioned between the transient and irreversible complexes in the ratios 3:1 for TEM-2 beta-lactamase and 1:1 for Klebsiella beta-lactamase. Biphasic inactivation was observed for both enzymes and the slower second phase was rate limited by the decay of the transiently stable complex. This decay released free enzyme for further reaction with fresh clavulanic acid, the products again partitioning between transiently stable and irreversibly inhibited enzyme. This cycle continued until all the enzyme had been irreversibly inhibited. A 115 molar excess of inhibitor was required to achieve complete inactivation of TEM-2 beta-lactamase. Hydrolysis of clavulanic acid with product release appeared to occur with the inhibition reaction, which explained this degree of clavulanic acid turnover. The stoichiometry of the interaction with Klebsiella beta-lactamase was not examined. The penicillinase from Proteus mirabilis C889 was rapidly inhibited by low concentrations of clavulanic acid. The major product was a moderately stable complex (t((1/2))=40min at pH7.3 and 37 degrees C); the proportion of the enzyme that was irreversibly inactivated was small. The cephalosporinase from Enterobacter cloacae P99 had low affinity for the inhibitor and only reacted with high concentrations of clavulanic acid (k=4.0m(-1).s(-1)) to produce a relatively stable complex (t((1/2))=180min at pH7.3 and 37 degrees C). No irreversible inactivation of this enzyme was detected. The rates of decay of the clavulanate-enzyme complexes produced in reactions with Proteus and Enterobacter enzymes were markedly increased at acid pH.

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The inhibition of staphylococcal beta-lactamase by clavulanic acid.

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