Anomalous pH dependence of the reactions of carbenicillin and sulbenicillin with Bacillus cereus .beta.-lactamase I. Influence of the .alpha.-substituent charge on the kinetic parameters

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Abstract
The pH dependence of kcat for the B. cereus .beta.-lactamase I catalyzed hydrolysis of carbenicillin (VI), which differs from benzylpenicillin (I) in having a carboxylic moiety .alpha. to the phenyl ring, exhibits a profile consistent with a model in which the .alpha.-COOH and .alpha.-COO- forms of the ES [enzyme-substrate] complex turn over with respective rate constants of 2152 s-1 and 384 s-1. the pKa(app) for the .alpha.-COOH is shifted from 3.2 in solution to 6.1 in th ES complex. The normalized kcat/Km vs. pH profile for VI is not superimposable on that of I, indicating that the neutral and anionic forms of the carboxyl moiety of VI combine with the enzyme to give the first irreversibly formed complex, presumably the acyl-enzyme. Quantitative accord with the kinetic data is achieved only through fitting to a model where kinetically significant proton transfer in the ES complex is permitted. The 2nd-order rate constants for the reaction of the enzyme with the .alpha.-COOH and .alpha.-COO- forms of VI are 2.2 .times. 108 and 3.8 .times. 106 M-1 s-1, respectively. The high value for the .alpha.-COOH form suggests that this reaction may be in part diffusion controlled. This conjecture is borne out by the observation that the sensitivity of kcat/Km to .eta.rel decreases with increasing pH for VI, whereas this sensitivity is pH independent for I. These conclusions are further supported by the results of a kinetic investigation of the pH dependence of sulbenicillin (VII) where an .alpha.-SO3H replaces the .alpha.-COOH of VI. The strongly acidic sulfonic acid moiety of VII is fully ionized throughout nearly the entire pH range of interest and its kinetics, as a function of pH, are very similar to those observed and calculated for the .alpha.-COO- form of VI. Solvent deuterium kinetic isotope effects are reported for kcat and kcat/Km for VI and VII.