Journal of Biological Chemistry
Volume 278, Issue 3, 17 January 2003, Pages 1848-1855
Journal home page for Journal of Biological Chemistry

ENZYME CATALYSIS AND REGULATION
An Extremely Potent Inhibitor of Xanthine Oxidoreductase: CRYSTAL STRUCTURE OF THE ENZYME-INHIBITOR COMPLEX AND MECHANISM OF INHIBITION*

https://doi.org/10.1074/jbc.M208307200Get rights and content
Under a Creative Commons license
open access

TEI-6720 (2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazolecarboxylic acid) is an extremely potent inhibitor of xanthine oxidoreductase. Steady state kinetics measurements exhibit mixed type inhibition withK i and K i′ values of 1.2 ± 0.05 × 10−10m and 9 ± 0.05 × 10−10m, respectively. Fluorescence-monitored titration experiments showed that TEI-6720 bound very tightly to both the active and the inactive desulfo-form of the enzyme. The dissociation constant determined for the desulfo-form was 2 ± 0.03 × 10−9m; for the active form, the corresponding number was too low to allow accurate measurements. The crystal structure of the active sulfo-form of milk xanthine dehydrogenase complexed with TEI-6720 and determined at 2.8-Å resolution revealed the inhibitor molecule bound in a long, narrow channel leading to the molybdenum-pterin active site of the enzyme. It filled up most of the channel and the immediate environment of the cofactor, very effectively inhibiting the activity of the enzyme through the prevention of substrate binding. Although the inhibitor did not directly coordinate to the molybdenum ion, numerous hydrogen bonds as well as hydrophobic interactions with the protein matrix were observed, most of which are also used in substrate recognition.

Cited by (0)

Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M208307200

*

This work was supported by Grants-in-aid 11169231 and 12147208 (to T. N.) for Science Research on Priority Areas, Grant-in-aid 13480212 (to T. N.) for Science Research from the Ministry of Education, Science, Sports and Culture of Japan, and a grant from the Canadian Institutes of Health Research, Institute of Genetics (to E. F. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

§

Both authors contributed equally to the results of this article.

§§

To whom correspondence and reprint requests may be addressed

¶¶

To whom correspondence and reprint requests may be addressed