Mechanism of the Conversion of Xanthine Dehydrogenase to Xanthine Oxidase

IDENTIFICATION OF THE TWO CYSTEINE DISULFIDE BONDS AND CRYSTAL STRUCTURE OF A NON-CONVERTIBLE RAT LIVER XANTHINE DEHYDROGENASE MUTANT*

  1. Takeshi Nishino
  1. Department of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan and Departments of Biochemistry, Medical Biophysics, and Molecular and Medical Genetics, Division of Molecular and Structural Biology, University of Toronto, Ontario Cancer Institute/University Health Network, Toronto, Ontario M5G 2M9, Canada
  1. To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan. Tel.: 81-3-3822-2131; Fax: 81-3-5685-3054; E-mail: nishino{at}nms.ac.jp.

Abstract

Mammalian xanthine dehydrogenase can be converted to xanthine oxidase by modification of cysteine residues or by proteolysis of the enzyme polypeptide chain. Here we present evidence that the Cys535 and Cys992 residues of rat liver enzyme are indeed involved in the rapid conversion from the dehydrogenase to the oxidase. The purified mutants C535A and/or C992R were significantly resistant to conversion by incubation with 4,4′-dithiodipyridine, whereas the recombinant wild-type enzyme converted readily to the oxidase type, indicating that these residues are responsible for the rapid conversion. The C535A/C992R mutant, however, converted very slowly during prolonged incubation with 4,4′-dithiodipyridine, and this slow conversion was blocked by the addition of NADH, suggesting that another cysteine couple located near the NAD+ binding site is responsible for the slower conversion. On the other hand, the C535A/C992R/C1316S and C535A/C992R/C1324S mutants were completely resistant to conversion, even on prolonged incubation with 4,4′-dithiodipyridine, indicating that Cys1316 and Cys1324 are responsible for the slow conversion. The crystal structure of the C535A/C992R/C1324S mutant was determined in its demolybdo form, confirming its dehydrogenase conformation.

  • Received February 17, 2005.
  • Revision received March 28, 2005.
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  1. The Journal of Biological Chemistry 280, 24888-24894.
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