Mutations at residue 244 (Ambler numbering system) in the class A TEM β-lactamase confer resistance to inactivation by β-lactamase inhibitors and result in diminished turnover of β-lactam substrates. The Arg 2 4 4 Ser mutant of the OHIO-1 β-lactamase, an SHV family enzyme, demonstrates variable susceptibilities to β-lactamase inhibitors and has significantly reduced catalytic efficiency. The minimum inhibitory concentrations (MICs) for Escherichia coli DH5α expressing the Arg 2 4 4 Ser β-lactamase were reduced when compared to the strain bearing the OHIO-1 β-lactamase: ampicillin, 512 vs. 8192 μg ml - 1 ; cephaloridine, 4 vs. 32 μg ml - 1 , respectively. The MICs for the β-lactam β-lactamase inhibitor combinations demonstrated resistance only to ampicillin-clavulanate, 16/8 vs. 8/4 μg ml - 1 respectively. In contrast, there was increased susceptibility to ampicillin-sulbactam, ampicillin-tazobactam, and piperacillin-tazobactam. When compared to the OHIO-1 β-lactamase homogenous preparations of the Arg 2 4 4 Ser β-lactamase enzyme demonstrated increased K m and decreased k c a t values for benzylpenicillin (K m =17 vs. 50 μM, k c a t =345 vs. 234 s - 1 ) and cephaloridine (K m =97 vs. 202 μM, k c a t =1023 vs. 202 s - 1 ). Although the K i and IC 5 0 values were increased for each inhibitor when compared to OHIO-1 β-lactamase, the turnover numbers (t n ) required for inactivation were increased only for clavulanate. For the Arg 2 4 4 Ser mutant enzyme of OHIO-1, the increased K i , decreased t n for the sulfones, and different partition ratio (k c a t /k i n a c t ) support the notion that not all class A enzymes are inactivated in the same manner, and that certain class A β-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.