OHIO-1 β-lactamase mutants:: the Arg244Ser mutant and resistance to β-lactams and β-lactamase inhibitors

Mutations at residue 244 (Ambler numbering system) in the class A TEM beta-lactamase confer resistance to inactivation by beta-lactamase inhibitors and result in diminished turnover of beta-lactam substrates. The Arg(244)Ser mutant of the OHIO-1 beta-lactamase, an SHV family enzyme, demonstrates variable susceptibilities to beta-lactamase inhibitors and has significantly reduced catalytic efficiency. The minimum inhibitory concentrations (MICs) for Escherichia coli DH5 alpha expressing the Arg(244)Ser beta-lactamase were reduced when compared to the strain bearing the OHIO-1 beta-lactamase: ampicillin, 512 vs. 8192 mu g ml(-1); cephaloridine, 4 vs, 32 mu g ml(-1), respectively. The MICs for the beta-lactam beta-lactamase inhibitor combinations demonstrated resistance only to ampicillin-clavulanate, 16/8 vs. 8/4 mu g ml(-1) respectively. In contrast, there was increased susceptibility to ampicillin-sulbactam, ampicillin-tazobactam, and piperacillin-tazobactam. When compared to the OHIO-1 beta-lactamase homogenous preparations of the Arg(244)Ser beta-lactamase enzyme demonstrated increased K-m and decreased k(cat) values for benzylpenicillin (K-m = 17 vs. 50 mu M, k(cat) = 345 vs. 234 s(-1)) and cephaloridine (K-m = 97 vs. 202 mu M, k(cat) = 1023 vs, 202 s(-1)). Although the K-i and IC50 values were increased for each inhibitor when compared to OHIO-I beta-lactamase, the turnover numbers (t(n)) required for inactivation were increased only for clavulanate. For the Arg(244)Ser mutant enzyme of OHIO-1, the increased K-i, decreased t(n) for the sulfones, and different partition ratio (k(cat)/k(inact)) support the notion that not all class A enzymes are inactivated in the same manner, and that certain class A beta-lactamase enzymes may react differently with identical substitutions in structurally conserved amino acids, The resistance phenotype of a specific mutations can vary depending on the enzyme. (C) 1999 Elsevier Science B,V, All rights reserved.