A DRAMATIC CHANGE IN THE RATE-LIMITING STEP OF BETA-LACTAM HYDROLYSISRESULTS FROM THE SUBSTITUTION OF THE ACTIVE-SITE SERINE RESIDUE BY A CYSTEINE IN THE CLASS-C BETA-LACTAMASE OF ENTEROBACTER-CLOACAE-908R

A cysteine residue has been substituted for the active-site serine of the class-C beta-lactamase produced by Enterobacter cloacae 908R by si te-directed mutagenesis. The modified protein exhibited drastically re duced k(cat.)/K(m) values on all tested substrates. However, this decr ease was due to increased K(m) values with some substrates and to decr eased k(cat.) values with others. These apparently contradictory resul ts could be explained by a selective influence of the mutation on the first-order rate constant characteristic of the acylation step, a hypo thesis which was confirmed by the absence of detectable acylenzyme acc umulation with all the tested substrates, with the sole exception of c efoxitin.