The Inhibitor Thiomandelic Acid Binds to Both Metal Ions in Metallo-β-lactamase and Induces Positive Cooperativity in Metal Binding

Abstract

Thiomandelic acid is a simple, broad spectrum, and reasonably potent inhibitor of metallo-␤-lactamases, enzymes that mediate resistance to ␤-lactam antibiotics. We report studies by NMR and perturbed angular correlation (PAC) spectroscopy of the mode of binding of the R and S enantiomers of thiomandelic acid, focusing on their interaction with the two metal ions in cadmium-substituted Bacillus cereus metallo-␤-lactamase. The 113 Cd resonances are specifically assigned to the metals in the two individual sites on the protein by using 113 Cd-edited 1 H NMR spectra. Each enantiomer of thiomandelate produces large downfield shifts of both 113 Cd resonances and changes in the PAC spectra, which indicate that they bind such that the thiol of the inhibitor bridges between the two metals. For R-thiomandelate, this is unambiguously confirmed by the observation of scalar coupling between H␣ of the inhibitor and both cadmium ions. The NMR and PAC spectra reveal that the two chiral forms of the inhibitor differ in the details of their coordination geometry. The complex with R-thiomandelate, but not that with the S-enantiomer, shows evidence in the PAC spectra of a dynamic process in the nanosecond time regime, the possible nature of which is discussed. The thiomandelate complex of the mononuclear enzyme can be detected only at low metal to enzyme stoichiometry; the relative populations of mononuclear and binuclear enzyme as a function of cadmium concentration provide clear evidence for positive cooperativity in metal ion binding in the presence of the inhibitor, in contrast to the negative cooperativity observed in the free enzyme.␤-Lactamases inactivate ␤-lactam antibiotics by catalyzing the hydrolysis of their endocyclic amide bond and play a major role in the emergence of strains of pathogenic bacteria resistant to these important antibiotics (1). The ␤-lactamases that contain a nucleophilic serine side chain as a key component of their active site (classes A, C, and D) have been extensively studied due to their established clinical importance. The class B ␤-lactamases or metallo-␤-lactamases (MBLs) 1 are metalloproteins that require one or two zinc ion(s) for their activity (2) and are less well understood. The first of these enzymes to be discovered was produced by an innocuous strain of Bacillus cereus (3), but in the last 20 years, MBL-mediated resistance has appeared in several pathogenic strains and is being rapidly spread by horizontal transfer, involving both plasmid-and integron-borne genetic elements (4, 5).The structures of several MBLs have been solved by x-ray diffraction and reveal two potential zinc ion binding sites at the active site (6 -16). The zinc ligands are not fully conserved between the different subclasses of MBL. In the subclass B1 enzymes, such as the B. cereus enzyme BcII, which is the subject of the present work, the zinc in site 1 is coordinated by the imidazole rings of three histidine residues (86, 88, and 149 in the B. cereus enzyme or 116, 118, and 196 using the class B ␤-lactamase (B...

Catalytic Role of the Metal Ion in the Metallo-β-lactamase GOB

“…bound to sites DCH and D3H, respectively, differing significantly from the resonance here reported (52,53). The 115 ppm resonance observed for 113 Cd(II)-GOB-18 agrees well with experimental 113 Cd NMR data giving a resonance at 120 ppm for carboxypeptidase A (54), presumably with a N 2 O 3 coordination sphere (two histidines, one water, and a bidentate carboxylate) and also with theoretical predictions (55).…”

Studies on ternary metallo-β lactamase-inhibitor complexes using electrospray ionization mass spectrometry

“…The results for thiomandelate with the CphA enzyme also provided unexpected new information on inhibition of the CphA enzyme. This inhibitor induced an increase of the affinity for binding of a second zinc ion in the complexed compared to uncomplexed enzyme, analogous to observations on the inhibition of the mono-cadmium form of the BcII enzyme [8], where thiomandelate induced conversion of the mono-to the dicadmium enzyme. Thus, the present work indicates that the differences in the stoichiometry of metal binding by the MBLs may be more subtle than previously appreciated.…”

“…The substrate profile of the family, coupled with the different metal stoichiometries, poses a challenge for the design of clinically useful inhibitors. Mercaptocarboxylates have been identified as competitive inhibitors of the MBLs [7] and structures of the MBLs reveal that the thiol group of inhibitor is chelated by both zinc ions [8,9].The aim of this work was to investigate the utility of ESI-MS for the screening of MBL inhibitors, by direct analysis of enzyme:metal:inhibitor complexes. Precedents for the use of ESI-MS to directly investigate…”

Metallo-?-lactamases: two binding sites for one catalytic metal ion?

“…A combined Cd 113 -NMR and PAC spectroscopic study of the enzyme at various cadmium/enzyme stoichiometries resulted in data which suggested that it was only possible to obtain the binuclear inhibited enzyme and not a mononuclear inhibited species. The inevitable conclusion of the authors was that the presence of thiomandelate induced positive cooperativity of cadmium binding for BcII [135]. The latter findings reinforce the question how many zinc ions are required and what is the physiologically relevant enzyme species to be considered as a target in inhibitor design.…”