title: 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.
authors.affiliation: Research Service, Department of Veterans Affairs Medical Center, Cleveland, OH.
authors.forename: R A
authors.initials: RA
authors.lastname: Bonomo
authors.forename: C G
authors.initials: CG
authors.lastname: Dawes
authors.forename: J R
authors.initials: JR
authors.lastname: Knox
authors.forename: D M
authors.initials: DM
authors.lastname: Shlaes
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry; D001483:Base Sequence; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011487:Protein Conformation; D014867:Water / Q000737:chemistry; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047090:beta-Lactams
chemical_list: D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D047090:beta-Lactams; D014867:Water; D001618:beta-Lactamases