Download MendeleyMechanism 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
Nishino, T., Okamoto, K., Kawaguchi, Y., Hori, H., Matsumura, T., Eger, B.T., Pai, E.F., Nishino, T.(2005) J Biol Chem 280: 24888-24894
- PubMed: 15878860
- DOI: https://doi.org/10.1074/jbc.M501830200
- Primary Citation of Related Structures:
1WYG - PubMed 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 Cys(535) and Cys(992) 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 Cys(1316) and Cys(1324) 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.
Organizational Affiliation:
Department of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan. [email protected]
