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  2. The inhibitor thiomandelic acid binds to both metal ions in metallo-beta-lactamase and induces positive cooperativity in metal binding

The inhibitor thiomandelic acid binds to both metal ions in metallo-beta-lactamase and induces positive cooperativity in metal binding

  • J Biol Chem. 2003 Aug 1;278(31):29240-51. doi: 10.1074/jbc.M301562200.
Christian Damblon 1 Mikael Jensen Abdessamad Ababou Igor Barsukov Cyril Papamicael Christopher J Schofield Lars Olsen Rogert Bauer Gordon C K Roberts
Affiliations

Affiliation

  • 1 Biological NMR Centre, Department of Biochemistry, University of Leicester, P.O. Box 138, University Road, Leicester LE1 9HN, United Kingdom.
Abstract

Thiomandelic acid is a simple, broad spectrum, and reasonably potent inhibitor of metallo-beta-lactamases, Enzymes that mediate resistance to Beta-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-beta-lactamase. The 113Cd resonances are specifically assigned to the metals in the two individual sites on the protein by using 113Cd-edited 1H NMR spectra. Each enantiomer of thiomandelate produces large downfield shifts of both 113Cd 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 Halpha 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.

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