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.
Highlights
Xanthine oxidoreductase (XOR)1 plays an important function in vertebrate metabolism by catalyzing the oxidation of xanthine to uric acid, the rate-limiting step in purine degradation [1]
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
It is well established that mammalian XOR can be reversibly converted from an NADϩ-dependent dehydrogenase to an O2dependent oxidase by oxidation of cysteine thiols to disulfide bonds [11, 12]
Summary
Materials—XOR (2.5–3.1 IU/mg) isolated from bovine milk in the oxidase form (XO) according to Waud et al [23] was a generous gift from Dr Joseph McCord (Webb-Waring Antioxidant Research Institute, University of Colorado Health Sciences Center). For one-dimensional electrophoresis samples were diluted with an equal volumes of 2ϫ concentrated SDS-PAGE sample buffer (0.125 M Tris, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, pH 6.5), heated for 3 min at 95 °C, and separated by electrophoresis in 10% acrylamide gels containing 0.1% SDS at room temperature. For twodimensional electrophoresis samples were diluted with equal volumes of 2ϫ concentrated SDS-PAGE sample buffer without 2-mercaptoethanol, heated for 3 min at 95 °C, and subjected to electrophoresis in 10% acrylamide gels as described above. When the tracking dye reached the bottom of the gel, electrophoresis was stopped and the protein-containing lanes were excised and prepared for electrophoresis in the second dimension by incubation in 3 ml of SDS-PAGE sample buffer containing 5% 2-mercaptoethanol for 1 h at 60 °C. The electrophoretic characteristics of the cross-linked protein were determined by SDS-PAGE analysis and silver staining as described above
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