Familial mutations and zinc stoichiometry determine the rate-limiting stepof nitrocefin hydrolysis by metallo-beta-lactamase from Bacteroides fragilis

The diverse members of the metallo-beta -lactamase family are a growing cli nical threat evolving under considerable selective pressure. The enzyme fro m Bacillus cereus differs from the Bacteroides fragilis enzyme in sequence, zinc stoichiometry, and mechanism. To chart the evolution of the more reac tive B. fragilis enzyme, we have made changes in an active site cysteine re sidue as well as in zinc content to mimic that which occurs in the B. cereu s enzyme. Specifically, by introducing a C104R mutation into the B. fragili s enzyme, binding of two zinc ions is maintained, but the k(cat) value for nitrocefin hydrolysis is decreased from 226 to 14 s(-1). Removal of 1 equiv of zinc from this mutant further decreases k(cat) to 4.4 s(-1). In both ca ses, the observed k(cat) closely approximates that found in the di- and mon ozinc forms of the B. cereus enzyme (12 and 6 s(-1), respectively). Pre-ste ady-state stopped-flow studies using nitrocefin as a substrate indicate tha t these enzyme forms share a similar mechanism featuring an anionic interme diate but that the rate-limiting step changes from protonation of that spec ies to the C-N bond cleavage leading to the intermediate. Overall, features that contribute 3.7 kcal/mol toward the acceleration of the C-N bond cleav age step have been uncovered although some of the total acceleration is mas ked in the steady-state by a change in rate-limiting step. These experiment s illustrate one step in the evolution of a catalytic mechanism and, in a l arger perspective, one step in the evolution of antibiotic resistance mecha nisms.