Inactivation of Bacillus cereus .beta.-lactamase I by 6.beta.-bromopenicillanic acid: kinetics

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Abstract
The kinetics of the inactivation of B. cereus .beta.-lactamase I by 6.beta.-bromopenicillanic acid are described. Loss of .beta.-lactamase activity is accompanied by a decrease in protein fluorescence, by the appearance of a protein-bound chromophore at 326 nm and by loss of tritium from 6.alpha.-[3H]-6.beta.-bromopenicillanic acid. All of the above changes probably have the same rate-determining step. The inactivation reaction is competitively inhibited by cephalosporin C, a competitive inhibitor of this enzyme, and by covalently bound clavulanic acid, suggesting that 6.beta.-bromopenicillanic acid reacts directly with the .beta.-lactamase active site. This inhibitor reacts initially as a normal substrate and that the rate-determining step of the inactivation is acylation of the enzyme. A rapid irreversible inactivation reaction rather than normal hydrolysis of the acyl-enzyme then follows acylation; 6.beta.-bromopenicillanic acid is thus a suicide substrate.