title: Structural and conformational analysis of the oxidase to dehydrogenase conversion of xanthine oxidoreductase.
abstract: 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.
authors.affiliation: Department of Physiology and Biophysics, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA. jim.mcmanaman@uchsc.edu
authors.forename: James L
authors.initials: JL
authors.lastname: McManaman
authors.forename: David L
authors.initials: DL
authors.lastname: Bain
mesh_terms: D000818:Animals; D002417:Cattle; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D015180:Electrophoresis, Gel, Two-Dimensional; D008239:Lysine / Q000737:chemistry; D008715:Methionine / Q000737:chemistry; D008856:Microscopy, Fluorescence; D010088:Oxidoreductases / Q000737:chemistry*; D010100:Oxygen / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology; D014968:Xanthine Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D014969:Xanthine Oxidase / Q000737:chemistry* / Q000502:physiology
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; D020033:Protein Isoforms; D013438:Sulfhydryl Compounds; D010649:Phenylalanine; D008715:Methionine; D010088:Oxidoreductases; D014968:Xanthine Dehydrogenase; D014969:Xanthine Oxidase; D002918:Chymotrypsin; D014357:Trypsin; D008239:Lysine; D003545:Cysteine; D010100:Oxygen