beta-Lactamase mutations far from the active site influence inhibitor binding.

Abstract

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 Asn276-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 Asn276-Gly mutant strain was more resistant to clavulanate (0.25 vs. 2.0 micrograms/ml) in the presence of ampicillin (16 micrograms/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 Ki of clavulanate for the Asn276-Gly enzyme (4.5 microM) was 10-fold greater than OHIO-1 (0.4 microM). For sulbactam and tazobactam the apparent Ki decreased for Asn276-Gly enzyme (1.0 and 0.1 micrograms/ml, respectively) compared to the wild-type parent (17 and 0.7 micrograms/ml, respectively). Comparing the Asn276-Gly heta-lactamase with OHIO-1, the Vmax for most substrates except cephaloridine did not change substantially. There was a 2-15 fold decreased affinity (Km) and catalytic efficiency (Vmax/Km) 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.

Cite this paper

@article{Bonomo1995betaLactamaseMF, title={beta-Lactamase mutations far from the active site influence inhibitor binding.}, author={Robert A. Bonomo and Christopher G. Dawes and James R Knox and David M. Shlaes}, journal={Biochimica et biophysica acta}, year={1995}, volume={1247 1}, pages={121-5} }