β-Lactamase mutations far from the active site influence inhibitor binding

Abstract

Analysis of the three dimensional structure of the class A β-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 μg/ml) in the presence of ampicillin (16 μg/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 β-lactams were tested. Consistent with the susceptibility test results, the apparent K i of clavulanate for the Asn 276-Gly enzyme (4.5 μM) was 10-fold greater than OHIO-1 (0.4 μM). For sulbactam and tazobactam the apparent K i decreased for Asn 276-Gly enzyme (1.0 and 0.1 μg/ml, respectively) compared to the wild-type parent (17 and 0.7 μg/ml, respectively). Comparing the Asn 276-Gly β-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 β-lactam substrates. These data support the observation and emphasize the role for this H bonding residue in orienting Arg-244 towards the active site.