COMPLEMENTARY ROLES OF MUTATIONS AT POSITION-69 AND POSITION-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 Met(69)-IleGly(242)-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. Met(69)-IleGly(242)Cys hydrolyzed cephaloridine (K-m = 213 mu M) but had K-m > 500 mu M for other beta-lactams tested including cefotaxime, and demonstrated a higher apparent K-i for inhibitors (clavulanate K-i = 500 mu M, sulbactam = 434 mu M, and tazobactam = 70 mu M). In a competition experiment with cephaloridine, the apparent K-i values for penicillin and cefotaxime remained low, 21 mu M and 0.7 mu M, 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 beta-lactam substrates or inhibitors.