title: Covalently bound phosphate residues in bovine milk xanthine oxidase and in glucose oxidase from Aspergillus niger: a reevaluation.
abstract: The reported presence of covalently bound phosphate residues in flavoproteins has significant implications with regard to the catalytic mechanisms and structural stability of the specific enzymes themselves and in terms of general cellular metabolic regulation. These considerations have led to a reevaluation of the presence of covalently bound phosphorus in the flavoproteins xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.1.3.22) and glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4). Milk xanthine oxidase purified by a procedure that includes anion-exchange chromatography is shown to contain three phosphate residues. All three are noncovalently associated with the protein, two with the FAD cofactor, and one with the molybdenum cofactor. Results of chemical analysis and 31P NMR spectroscopy indicate that enzyme purified by this method contains no phosphoserine residues. Xanthine oxidase preparations purified by chromatography on calcium phosphate gel in place of DEAE-Sephadex yielded higher phosphate-to-protein ratios, which could be reduced to the expected values by additional purification on a folate affinity column. Highly active, highly purified preparations of glucose oxidase are shown to contain only the two phosphate residues of the FAD cofactor. The covalently bound bridging phosphate reported by others may arise in aged or degraded preparations of the enzyme but appears not to be a constituent of functional glucose oxidase. These results suggest that the presence of covalent phosphate residues in other flavoproteins should be rigorously reevaluated as well.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, NC 27710.
authors.forename: J L
authors.initials: JL
authors.lastname: Johnson
authors.forename: R E
authors.initials: RE
authors.lastname: London
authors.forename: K V
authors.initials: KV
authors.lastname: Rajagopalan
mesh_terms: D000818:Animals; D001234:Aspergillus niger / Q000201:enzymology*; D002417:Cattle; D005260:Female; D005420:Flavoproteins; D005949:Glucose Oxidase; D009682:Magnetic Resonance Spectroscopy; D008892:Milk / Q000201:enzymology*; D010710:Phosphates / Q000032:analysis*; D010758:Phosphorus; D010768:Phosphoserine / Q000032:analysis*; D012694:Serine / Q000031:analogs & derivatives*; D014969:Xanthine Oxidase / Q000302:isolation & purification
chemical_list: D005420:Flavoproteins; D010710:Phosphates; D010768:Phosphoserine; D010758:Phosphorus; D012694:Serine; D005949:Glucose Oxidase; D014969:Xanthine Oxidase