Complementary roles of mutations at positions 69 and 242 in a class A beta-lactamase.

Analysis of the three-dimensional structure of class A beta-lactamases suggests that deformation of the substrate binding site can be produced by changes in the hydrophobicity of residue 69 behind the beta-sheet and by outward movement of the B3 beta-strand by introduction of a non-glycine residue at position 242 on the B4 beta-strand. By site-directed mutagenesis Met69-IleGly242-Cys, a double mutant, of the OHIO-1 beta-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 beta-lactams. Met69-IleGly242Cys hydrolyzed cephaloridine (Km = 213 microM) but had Km > 500 microM for other beta-lactams tested including cefotaxime, and demonstrated a higher apparent Ki for inhibitors (clavulanate Ki = 500 microM sulbactam = 434 microM, and tazobactam = 70 microM). In a competition experiment with cephaloridine, the apparent Ki values for penicillin and cefotaxime remained low, 21 microM and 0.7 microM, respectively. Since Ile is twice as hydrophobic as Met, the Met69-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 Gly242-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 beta-lactam substrates or inhibitors.