Thiols as Classical and Slow-Binding Inhibitors of IMP-1 and Other Binuclear Metallo-β-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.

Metallo-β-Lactamase Detection: Comparative Evaluation of Double-Disk Synergy versus Combined Disk Tests for IMP-, GIM-, SIM-, SPM-, or VIM-Producing Isolates

“…In contrast, neutral IMBLs have a pronounced lag prior to the start of inhibitory activity; i.e., the best inhibitory results are achieved after preincubating the IMBL with MBL before adding the substrate. In this manner, the weakest activity of MET in this study could be explained by the absence of the preincubation step in the performance of the phenotypic detection (28).…”

Metallo-β-Lactamases: the Quiet before the Storm?

“…A variety of structurally disparate compounds have been examined as MBL inhibitors, including thioester derivatives (44,58,118,119,121), trifluoromethyl alcohols and ketones (182), thiols (6,18,44,56,57,71,76,101,155,159), sulfonyl hydrazones (160), tricyclic natural products (122), succinic acid derivatives (171), biphenyl tetrazoles (169,170), cysteinyl peptides (17), mercaptocarboxylates (57, 121), 1-␤-methylcarbapenem (104, 105) cefotetan (140), thioxocephalosporins (173), and penicillin derivatives (25). The activities of these compounds against selected MBLs are summarized in Table 5.…”

The Cysteine Dioxygenase Homologue from Pseudomonas aeruginosa Is a 3-Mercaptopropionate Dioxygenase

“…In one bacterium, Variovarax paradoxus, 3-MPA utilization was shown to involve dioxygenation of 3-MPA to 3-sulfinopropionic acid (3-SPA), catalyzed by a CDO homologue, 3-mercaptopropionate dioxygenase (3MDO), which showed high specificity for 3-MPA but not for cysteine (6). Interestingly, 3-MPA acts as an inhibitor of metallo-␤-lactamase, an enzyme involved in antibiotic resistance in bacteria (34,35). In mammals, 3-MPA is toxic by inhibiting the oxidation of fatty acid (36,37) and the synthesis of the inhibitory neurotransmitter ␥-aminobutyric acid, resulting in deformation, seizures, or death of rats (38 -40).…”