Analysis of the three-dimensional structure of class A β-lactamases suggests that deformation of the substrate binding site can be produced by changes in the hydrophobicity of residue 69 behind the β-sheet and by outward movement of the B3 β-strand by introduction of a non-glycine residue at position 242 on the B4 β-strand. By site-directed mutagenesis Met 69-IleGly 242-Cys, a double mutant, of the OHIO-1 β-lactamase, was constructed. The minimum inhibitory concentrations (MICs) of the double mutant compared with the wild type and each single mutant revealed an increased susceptibility to β-lactams. Met 69-IleGly 242Cys hydrolyzed cephaloridine ( K m = 213 μM) but had K m > 500 μM for other β-lactams tested including cefotaxime, and demonstrated a higher apparent K i for inhibitors (clavulanate K i = 500 μM, sulbactam = 434 μM, and tazobactam = 70 μM). In a competition experiment with cephaloridine, the apparent K i values for penicillin and cefotaxime remained low, 21 μM and 0.7 μM, respectively. Since Ile is twice as hydrophobic as Met, the Met 69-Ile mutation may result in partial collapse of the oxyanion hole. This would also increase the distance between Arg- 244 and the carboxyl of clavulanic acid. The Gly 242-Cys mutation opens the lower portion of the active site to bulky R groups of cephalosporins. Although these two mutations result in a catalytically impaired enzyme, they can be used to model the complementary role of two distinct residues, neither of which interacts directly with β-lactam substrates or inhibitors.