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J. Biol. Chem., Vol. 278, Issue 31, 29240-29251, August 1, 2003

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

Christian Damblon , Mikael Jensen , Abdessamad Ababou , Igor Barsukov , Cyril Papamicael ¶, Christopher J. Schofield ¶, Lars Olsen , Rogert Bauer  and Gordon C. K. Roberts  ||

From the Biological NMR Centre, Department of Biochemistry, University of Leicester, P.O. Box 138, University Road, Leicester LE1 9HN, United Kingdom, the Department of Mathematics and Physics, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark, and ^¶The Oxford Centre for Molecular Sciences and The Dyson Perrins Laboratory, South Parks Road, Oxford OX1 3QY, United Kingdom

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 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 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.

Received for publication, February 13, 2003 , and in revised form, April 28, 2003.

^* This work was supported by Biotechnology and Biological Sciences Research Council Grant 91/BI3539, by European Union TMR Network Grant CT 98-0232, and by the Danish Research Council for Natural Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains an additional figure.

^|| To whom correspondence should be addressed. Tel.: 44-116-252-2978; Fax: 44-116-223-1503; E-mail gcr{at}le.ac.uk.

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