title: Importance of position 170 in the inhibition of GES-type β-lactamases by clavulanic acid.
abstract: Bacterial resistance to β-lactam antibiotics (penicillins, cephalosporins, carbapenems, etc.) is commonly the result of the production of β-lactamases. The emergence of β-lactamases capable of turning over carbapenem antibiotics is of great concern, since these are often considered the last resort antibiotics in the treatment of life-threatening infections. β-Lactamases of the GES family are extended-spectrum enzymes that include members that have acquired carbapenemase activity through a single amino acid substitution at position 170. We investigated inhibition of the GES-1, -2, and -5 β-lactamases by the clinically important β-lactamase inhibitor clavulanic acid. While GES-1 and -5 are susceptible to inhibition by clavulanic acid, GES-2 shows the greatest susceptibility. This is the only variant to possess the canonical asparagine at position 170. The enzyme with asparagine, as opposed to glycine (GES-1) or serine (GES-5), then leads to a higher affinity for clavulanic acid (K(i) = 5 μM), a higher rate constant for inhibition, and a lower partition ratio (r ≈ 20). Asparagine at position 170 also results in the formation of stable complexes, such as a cross-linked species and a hydrated aldehyde. In contrast, serine at position 170 leads to formation of a long-lived trans-enamine species. These studies provide new insight into the importance of the residue at position 170 in determining the susceptibility of GES enzymes to clavulanic acid.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
authors.forename: Hilary
authors.initials: H
authors.lastname: Frase
authors.forename: Marta
authors.initials: M
authors.lastname: Toth
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Champion
authors.forename: Nuno T
authors.initials: NT
authors.lastname: Antunes
authors.forename: Sergei B
authors.initials: SB
authors.lastname: Vakulenko
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001216:Asparagine / Q000235:genetics; D002853:Chromatography, Liquid; D019818:Clavulanic Acid / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005998:Glycine / Q000235:genetics; D008826:Microbial Sensitivity Tests; D012694:Serine / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D013329:Structure-Activity Relationship; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; D019818:Clavulanic Acid; D012694:Serine; D001216:Asparagine; D001618:beta-Lactamases; D005998:Glycine