Determination of partition ratios for allylisopropylacetamide during suicidal processing by a phenobarbital-induced cytochrome P-450 isozyme from rat liver.

Allylisopropylacetamide is shown to be a suicide substrate for the phenobarbital-inducible cytochromes P-450. In phenobarbital-induced rat liver microsomes about 70% of the cytochrome P-450-mediated N,N-dimethylaniline N-demethylase activity is sensitive to allylisopropylacetamide inactivation; the residual 30% of the N-demethylase activity is incapable of allylisopropylacetamide turnover and insensitive to allylisopropylacetamide inactivation. The partition number for inactivation of the susceptible population of cytochrome P-450 indicates turnover of 201 molecules of allylisopropylacetamide per molecule of P-450 inactivated. A purified phenobarbital-induced isozyme of cytochrome P-450, when reconstituted with purified rat liver cytochrome P-450 reductase, is also inactivated by allylisopropylacetamide in a suicide fashion with a corrected partition ratio of 184 turnovers of allylisopropylacetamide per inactivation event. This partition number is corrected for the competing O2-dependent autoinactivation of cytochrome P-450 which we have previously shown to occur with the purified isozyme (Loosemore, M., Light, D. R., and W#alsh, C. (1980) J. Biol. Chem. 255, 9017-9020). The 201 product molecules of cytochrome P-450-mediated turnover of allylisopropylacetamide in either the microsomal or purified enzyme system are probably the epoxide, are reactive toward alkylation of cellular nucleophiles, and covalently modify protein and exogenous calf thymus DNA molecules.

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

The kinetics of the inactivation of Bacillus 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. It is shown that 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. It is proposed that 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.

6-beta-bromopenicillanic acid, a potent beta-lactamase inhibitor.

6-beta-Bromopenicillanic acid, which arises from the epimerization of 6-alpha-bromopenicillanic acid in aqueous solution or from hydrogenation of 6,6-dibromopenicillanic acid, is a powerful, irreversible, active-site-directed inhibitor of several typical beta-lactamases (penicillinase; penicillin amido-beta-lactamhydrolase, EC 3.5.2.6); 6-alpha-bromopenicillanic acid, being completely inhibited at less than micromolar concentrations through what is probably a 1:1 interaction. The B. licheniformis exoenzyme is similarly susceptible, while the Staphylococcus aureus enzyme and the Escherichia coli (R factor) enzyme are less so; the B. cereus beta-lactamase II is not inhibited. Very high concentrations (greater than or equal to 0.1 M) of benzylpenicillin, a good substrate, are required to significantly reduce the rate of inhibition of B. cereus beta-lactamase I by 6-beta-bromopenicillanic acid.