BETA-LACTAMASE MUTATIONS FAR FROM THE ACTIVE-SITE INFLUENCE INHIBITORBINDING

Analysis of the three dimensional structure of the class A beta-lactam ases shows that Arg-244, a spatially conserved residue important for i nactivation by clavulanic acid, is held in place by a hydrogen (H) bon d from the residue at 276. An Asn(276)-Gly mutant of OHIO-1, an SHV fa mily 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 amp icillin as the OHIO-1 bearing strain. No difference in MICs was observ ed when other beta-lactams were tested. Consistent with the susceptibi lity test results, the apparent K-i of clavulanate for the Asn(276)-Gl y enzyme (4.5 mu M) was 10-fold greater than OHIO-1 (0.4 mu M). For su lbactam and tazobactam the apparent K-i decreased for Asn(276)-Gly enz yme (1.0 and 0.1 mu g/ml, respectively) compared to the wild-type pare nt (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 cephalori dine did not change substantially. There was a 2-15 fold decreased aff inity (K-m) and catalytic efficiency (V-max/K-m) for beta-lactam subst rates. These data support the observation and emphasize the role for t his H bonding residue in orienting Arg-244 towards the active site.