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Originally published In Press as doi:10.1074/jbc.M200828200 on March 25, 2002

J. Biol. Chem., Vol. 277, Issue 24, 21261-21268, June 14, 2002
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Structural and Conformational Analysis of the Oxidase to Dehydrogenase Conversion of Xanthine Oxidoreductase*

James L. McManamanDagger § and David L. Bain

From the Dagger  Department of Physiology and Biophysics and  Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, Colorado 80262

Xanthine oxidoreductase (XOR) is a 300-kDa homodimer that can exist as an NAD+-dependent dehydrogenase (XD) or as an O2-dependent oxidase (XO) depending on the oxidation state of its cysteine thiols. Both XD and XO undergo limited cleavage by chymotrypsin and trypsin. Trypsin selectively cleaved both enzyme forms at Lys184, while chymotrypsin cleaved XD primarily at Met181 but cleaved XO at Met181 and at Phe560. Chymotrypsin, but not trypsin, cleavage also prevented the reductive conversion of XO to XD; thus the region surrounding Phe560 appears to be important in the interconversion of the two forms. Size exclusion chromatography showed that disulfide bond formation reduced the hydrodynamic volume of the enzyme, and two-dimensional gel electrophoresis of chymotrypsin-digested XO showed significant, disulfide bond-mediated, conformational heterogeneity in the N-terminal third of the enzyme but no evidence of disulfide bonds between the N-terminal and C-terminal regions or between XOR subunits. These results indicate that intrasubunit disulfide bond formation leads to a global conformational change in XOR that results in the exposure of the region surrounding Phe560. Conformational changes within this region in turn appear to play a critical role in the interconversion between the XD and XO forms of the enzyme.

* This work was supported by National Institutes of Health Grants HL 45582-05AZ and P01CHD38129. Microsequencing was performed at the UCHSC Cancer Center's Protein Chemistry Core Laboratory supported by Grant CA46934 from the NCI, National Institutes of Health.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.

§ To whom correspondence should be addressed: Dept. of Physiology and Biophysics, University of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262. Tel.: 303-315-7093; Fax: 303-315-8110; E-mail: jim.mcmanaman@uchsc.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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