Kinetics of inactivation of β-lactamase I by 6 β-bromopenicillanic acid

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Abstract
The kinetics of the inactivation of .beta.-lactamase I from Bacillus cereus 569 by preparations of 6 .alpha.-bromopenicillanic acid showed unexpected features. These can be quantitatively accounted for on the basis of the inactivator being the epimer, 6.beta.-bromopenicillanic acid. At pH 9.2, the rate-determining step in the inactivation is the formation of the inactivator. When pure 6.beta.-bromopenicillanic acid is used to inactivate .beta.-lactamase I, simple 2nd order kinetics are observed. The inactivated enzyme has a new absorption peak at 326 nm. The rate constant for inactivation has the same value as the rate constant for appearance of absorption at 326 nm; the rate-determining step may thus be fission of the .beta.-lactam ring of 6.beta.-bromopenicillanic acid. Inactivation is slower in the presence of substrate, and the observed kinetics can be quantitatively accounted for on a simple competitive model. Inactivation is apparently a consequence of reaction at the active site.