Kinetic analysis of a catalytically‐compromised mutant of the class D β‐lactamase OXA‐1

The presence of β‐lactamase enzymes is the most important mechanism by which bacteria have gained resistance to lactam antibiotics such as penicillins and cephalosporins. The mechanism by which the Class D β‐lactamase OXA‐1 disables these drugs involves both an acylation and hydrolytic deacylation step. It has been proposed that the conserved residue Ser 115 may contribute to protonation of the nitrogen atom within the lactam ring encouraging bond breakage. Here, using site‐directed mutagenesis and steady‐state kinetic analysis, we have investigated whether or not Ser 115 acts as proton shuttle contributing to both the acylation and deacylation steps. In comparison to wildtype, we have determined that the Ser115Gly mutant has a significantly elevated K m and a greatly decreased K cat in the turnover of the penicillin ampicillin and cephalosporin cephalothin. Furthermore, subsequent rate measurements indicate that for both drugs, the acylation step is approximately six times as fast as the deacylation step. These results are consistent with the notion that Ser115 does play a critical catalytic role in the mechanism of the OXA‐1 β‐lactamase.