title: Thiols as classical and slow-binding inhibitors of IMP-1 and other binuclear metallo-beta-lactamases.
abstract: The inhibitory effect of a variety of thiol compounds on the function of binuclear metallo-beta-lactamases, with a particular focus on IMP-1 from Pseudomonas aeruginosa, has been investigated. Thiol inhibitors, depending on their structural features, fall into two categories, one in which inhibition at neutral pH was instantaneous and the other in which inhibition was time-dependent. While mercaptans with anionic substituents in the vicinity of their SH groups exhibited the former type of inhibition, neutral thiols appear to induce a slow, time-dependent isomerization of the initially formed EI complex to a tighter EI complex. Kinetic parameters describing the latter process were obtained by fitting progress curves of substrate hydrolysis using standard and numerical procedures. The failure of charged thiols to exhibit slow binding is suggested to be due to a rapid isomerization of the initial EI complex. Slow binding in the case of neutral thiols was observed only below pH 8. Studies on the pH dependence of catalysis by IMP-1 revealed that (i) enzyme inactivation at low pH is a slow process with presumably two groups with a pK(a) of approximately 5.2 in the protein being responsible for the loss of activity, (ii) inhibition by thiols is independent of pH between pH 5 and 9, and (iii) an apparent enhancement of the catalytic activity of IMP-1 by thiols occurs at pH <5. The last mentioned phenomenon is explained by a model in which mercaptans retard the proton-dependent isomerization of the enzyme. Studies on the thiol-mediated inhibition of the binuclear forms of Bacteroides fragilis (CcrA) and Bacillus cereus (BcII strain 5/B/6) metallo-beta-lactamase have revealed that while CcrA was instantaneously albeit moderately inhibited by mercaptans, BcII mimicked IMP-1 in its interaction with thiols. These differences are proposed to be due partly to the structural divergence of these proteins in the vicinity of Zn2.
authors.affiliation: Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
authors.forename: Stefan
authors.initials: S
authors.lastname: Siemann
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Clarke
authors.forename: Thammaiah
authors.initials: T
authors.lastname: Viswanatha
authors.forename: Gary I
authors.initials: GI
authors.lastname: Dmitrienko
mesh_terms: D001409:Bacillus cereus / Q000201:enzymology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D001667:Binding, Competitive; D002384:Catalysis / Q000187:drug effects; D002511:Cephalosporins / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008667:Metalloproteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D021241:Spectrometry, Mass, Electrospray Ionization; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D002511:Cephalosporins; D004791:Enzyme Inhibitors; D008667:Metalloproteins; D013438:Sulfhydryl Compounds; D065093:beta-Lactamase Inhibitors; C409201:beta-lactamase IMP-1; D001618:beta-Lactamases; C063912:carbapenemase; C021720:nitrocefin