BETA-LACTAMASE MUTATIONS FAR FROM THE ACTIVE-SITE INFLUENCE INHIBITOR BINDING

Analysis of the three dimensional structure of the class A beta-lactamases shows that Arg-244, a spatially conserved residue important for inactivation by clavulanic acid, is held in place by a hydrogen (H) bond from the residue at 276. An Asn(276)-Gly mutant of OHIO-1, an SHV family class A enzyme, was constructed to investigate the importance of that interaction. Compared to a strain expressing the wild type enzyme, OHIO-1, the MIC of the Asn(276)-Gly mutant strain was more resistant to clavulanate (0.25 vs. 2.0 mu g/ml) in the presence of ampicillin (16 mu g/ml) but was as susceptible to sulbactam or tazobactam plus ampicillin as the OHIO-1 bearing strain. No difference in MICs was observed when other beta-lactams were tested. Consistent with the susceptibility test results, the apparent K-i of clavulanate for the Asn(276)-Gly enzyme (4.5 mu M) was 10-fold greater than OHIO-1 (0.4 mu M). For sulbactam and tazobactam the apparent K-i decreased for Asn(276)-Gly enzyme (1.0 and 0.1 mu g/ml, respectively) compared to the wild-type parent (17 and 0.7 mu g/ml, respectively). Comparing the Asn(276)-Gly beta-lactamase with OHIO-1, the V-max for most substrates except cephaloridine did not change substantially. There was a 2-15 fold decreased affinity (K-m) and catalytic efficiency (V-max/K-m) for beta-lactam substrates. These data support the observation and emphasize the role for this H bonding residue in orienting Arg-244 towards the active site.