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Query: UNIPROT:P47989 (xanthine oxidase)
 8,633 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The rates of NADH oxidation in presence of xanthine oxidase increase to a small and variable extent on addition of high concentrations of lactate dehydrogenase and other dehydrogenases. This heat stable activity is similar to polyvanadate-stimulation with respect to pH profile and SOD sensitivity. Isocitric dehydrogenase (NADP-specific) showed heat labile, SOD-sensitive polyvanadate-stimulated NADH oxidation activity. Polyvanadate-stimulated SOD-sensitive NADH oxidation was also found to occur with riboflavin, FMN and FAD in presence of a non-specific protein, BSA, suggesting that some flavoproteins may possess this activity.
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PMID:Stimulation of NADH oxidation by xanthine oxidase and polyvanadate in presence of some dehydrogenases and flavin compounds. 178 72

31P ENDOR spectra are described for three different molybdenum(V) species in reduced xanthine oxidase samples. The spectra were not affected by removing the FAD from the enzyme, implying that this is located at some distance from molybdenum. Furthermore, in confirmation of the work of J. L. Johnson, R. E. London, and K. V. Rajagopalan [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6493-6497], NMR and chemical analysis of the phosphate content of highly purified xanthine oxidase showed there are only three phosphate residues per subunit of the enzyme. It is concluded that the ENDOR features are due to hyperfine coupling of the phosphate group of the pterin cofactor to the molybdenum atom. Evaluation of the dipolar component of the coupling has permitted estimation of the molybdenum-phosphorus distances as 7-12 A. This implies that the cofactor is in an extended conformation in the enzyme molecule. Less detailed 31P ENDOR data on sulfite oxidase are consistent with a similar conformation for the cofactor in this enzyme.
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PMID:31P ENDOR studies of xanthine oxidase: coupling of phosphorus of the pterin cofactor to molybdenum (V). 185 Feb 96

The steady-state and rapid kinetic properties of xanthine oxidase containing a series of FAD analogs of varying reduction potential have been investigated. From steady-state analysis, Vmax is found to exhibit a sigmoidal dependence on the flavin midpoint potential in the homologous series. This dependence is accurately described by a model in which the rate of catalysis is attenuated by the amount of partially reduced enzyme generated during turnover possessing an unfavorable distribution of reducing equivalents among the several redox-active centers of the protein. The model assumes that reducing equivalents equilibrate among these centers rapidly compared to the limiting rates for the reductive and oxidative half-reactions. This assumption is borne out by a quantitative analysis of the reductive and oxidative half-reactions of the several enzyme forms investigated in detail. It is demonstrated in these studies that xanthine oxidase containing low potential flavin derivatives such as 1-deaza, 6-hydroxy, or 8-hydroxy FAD exhibits low turnover not because of inherently slow rates of reduction by xanthine or oxidation by molecular oxygen, but because in partially reduced enzyme generated in the course of turnover reducing equivalents are distributed within the enzyme in such a way that the enzyme can participate in neither the reductive nor oxidative half-reactions. These results provide confirmation of the operation of a thermodynamic control mechanism in a simple electron-transferring system.
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PMID:The kinetic behavior of xanthine oxidase containing chemically modified flavins. 189 27

Electron transfer within milk xanthine oxidase has been examined by the technique of pulse radiolysis. Radiolytically generated N-methylnicotinamide radical or 5-deazalumiflavin radical has been used to rapidly and selectively introduce reducing equivalents into the enzyme so that subsequent equilibration among the four redox-active centers of the enzyme (a molybdenum center, two iron-sulfur centers, and FAD) could be monitored spectrophotometrically. Experiments have been performed at pH 6 and 8.5, and a comprehensive scheme describing electron equilibration within the enzyme at both pH values has been developed. All rate constants ascribed to equilibration between specific pairs of centers in the enzyme are found to be rapid relative to enzyme turnover under the same conditions. Electron equilibration between the molybdenum center and one of the iron-sulfur centers of the enzyme (tentatively assigned Fe/S I) is particularly rapid, with a pH-independent first-order rate constant of approximately 8.5 x 10(3) s-1. The results unambiguously demonstrate the role of the iron-sulfur centers of xanthine oxidase in mediating electron transfer between the molybdenum and flavin centers of the enzyme.
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PMID:Electron transfer in milk xanthine oxidase as studied by pulse radiolysis. 200

A comparative study using laser flash photolysis of the kinetics of reduction and intramolecular electron transfer among the redox centers of chicken liver xanthine dehydrogenase and of bovine milk xanthine oxidase is described. The photogenerated reductant, 5-deazariboflavin semiquinone, reacts with the dehydrogenase (presumably at the Mo center) in a second-order manner, with a rate constant (k = 6 x 10(7) M-1 s-1) similar to that observed with the oxidase [k = 3 x 10(7) M-1 s-1; Bhattacharyya et al. (1983) Biochemistry 22, 5270-5279]. In the case of the dehydrogenase, neutral FAD radical formation is found to occur by intramolecular electron transfer (kobs = 1600 s-1), presumably from the Mo center, whereas with the oxidase the flavin radical forms via a bimolecular process involving direct reduction by the deazaflavin semiquinone (k = 2 x 10(8) M-1 s-1). Biphasic rates of Fe/S center reduction are observed with both enzymes, which are due to intramolecular electron transfer (kobs approximately 100 s-1 and kobs = 8-11 s-1). Intramolecular oxidation of the FAD radical in each enzyme occurs with a rate constant comparable to that of the rapid phase of Fe/S center reduction. The methylviologen radical, generated by the reaction of the oxidized viologen with 5-deazariboflavin semiquinone, reacts with both the dehydrogenase and the oxidase in a second-order manner (k = 7 x 10(5) M-1 s-1 and 4 x 10(6) M-1 s-1, respectively). Alkylation of the FAD centers results in substantial alterations in the kinetics of the reaction of the viologen radical with the oxidase but not with the dehydrogenase. These results suggest that the viologen radical reacts directly with the FAD center in the oxidase but not in the dehydrogenase, as is the case with the deazaflavin radical. The data support the conclusion that the environments of the FAD centers differ in the two enzymes, which is in accord with other studies addressing this problem from a different perspective [Massey et al. (1989) J. Biol. Chem. 264, 10567-10573]. In contrast, the rate constants for intramolecular electron transfer among the Mo, FAD, and Fe/S centers in the two enzymes (where they can be determined) are quite similar.
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PMID:Kinetic comparison of reduction and intramolecular electron transfer in milk xanthine oxidase and chicken liver xanthine dehydrogenase by laser flash photolysis. 204 32

The cytosolic molybdoflavoprotein xanthine oxidase has been shown to catalyze the reduction of exocyclic nitro groups to the corresponding nitroso, hydroxylamino and amino derivatives for a wide variety of xenobiotics including the nitrated polycyclic aromatic hydrocarbons 1-nitropyrene and 3-nitrofluoranthene. Using commercially available bovine liver xanthine oxidase, we have studied the kinetics of the metabolism of 1-nitropyrene and 3-nitrofluoranthene. The nitroreduction of these nitro compounds in the presence of xanthine oxidase is dependent on the presence of hypoxanthine or xanthine and the absence of oxygen. This nitroreduction is independent of added flavins (FMN and FAD), unlike the related molybdoflavoprotein aldehyde oxidase. Xanthine oxidase has a Km of 0.7 microM and Vmax of 0.06 nmol/min per unit enzyme for 1-nitropyrene and a Km of 8.6 microM and Vmax of 0.7 nmol/min per unit enzyme for 3-nitrofluoranthene. The importance of these kinetic constants in evaluating the cytosolic metabolism of 1-nitropyrene and 3-nitrofluoranthene are discussed.
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PMID:The kinetics of 1-nitropyrene and 3-nitrofluoranthene metabolism using bovine liver xanthine oxidase. 220 87

Bovine milk xanthine oxidase was potently inhibited by 6-(bromomethyl)-9H-purine in a time-dependent process with O2 as the electron acceptor. If the enzyme were assayed with phenazene ethosulfate as an electron acceptor, 6-(bromomethyl)-9H-purine was not an inhibitor. The rate of formation of inhibited enzyme increased with increasing concentrations of 6-(halomethyl)-9H-purine, decreased with increasing concentrations of O2, and increased in the presence of xanthine. The inhibited enzyme regained activity nonactinically at pH 7 with a t1/2 of 31 h. The optical difference spectrum between native enzyme and inhibited enzyme suggested that the enzyme-bound FAD was modified. This conclusion was confirmed by demonstrating that activity was restored to the inhibited enzyme if the enzyme-bound flavin was removed by treatment with CaCl2 and the resulting apoenzyme was reconstituted with FAD. Aerobically, 6-(bromomethyl)-9H-purine was oxidized by the enzyme to a species having a UV spectrum consistent with hydroxylation of the purine ring to form a urate analogue. Anaerobically, the enzyme reduced 6-(bromomethyl)-9H-purine to 6-methylpurine with 1 mol of enzyme being completely inhibited after reduction of 23 mol of 6-(bromomethyl)-9H-purine. Thus, 6-(bromomethyl)-9H-purine was not only oxidized by xanthine oxidase but was also reduced by the enzyme in a reaction that partitioned between formation of 6-methylpurine and inhibition of the enzyme by modification of the enzyme-bound flavin. Similar results were found when 6-(chloromethyl)-9H-purine was the inhibitor.
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PMID:Xanthine oxidase-catalyzed reductive debromination of 6-(bromomethyl)-9H-purine with concomitant covalent modification of the FAD prosthetic group. 238 Jan 73

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.
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PMID:Covalently bound phosphate residues in bovine milk xanthine oxidase and in glucose oxidase from Aspergillus niger: a reevaluation. 250 51

The native flavin, FAD, was removed from chicken liver xanthine dehydrogenase and milk xanthine oxidase by incubation with CaCl2. The deflavoenzymes, still retaining their molybdopterin and iron-sulfur prosthetic groups, were reconstituted with a series of FAD derivatives containing chemically reactive or environmentally sensitive substituents in the isoalloxazine ring system. The reconstituted enzymes containing these artificial flavins were all catalytically active. With both the chicken liver dehydrogenase and the milk oxidase, the flavin 8-position was found to be freely accessible to solvent. The flavin 6-position was also freely accessible to solvent in milk xanthine oxidase, but was significantly less exposed to solvent in the chicken liver dehydrogenase. Pronounced differences in protein structure surrounding the bound flavin were indicated by the spectral properties of the two enzymes reconstituted with flavins containing ionizable -OH or -SH substituents at the flavin 6- or 8-positions. Milk xanthine oxidase either displayed no preference for binding of the neutral or anionic flavin (8-OH-FAD) or a slight preference for the anionic form of the flavin (6-hydroxy-FAD, 6-mercapto-FAD, and possibly 8-mercapto-FAD). On the other hand, the chicken liver dehydrogenase had a dramatic preference for binding the neutral (protonated) forms of all four flavins, perturbing the pK of the ionizable substituent greater than or equal to 4 pH units. These results imply the existence of a strong negative charge in the flavin binding site of the dehydrogenase, which is absent in the oxidase.
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PMID:Differences in protein structure of xanthine dehydrogenase and xanthine oxidase revealed by reconstitution with flavin active site probes. 273 38

Methylene blue competes 100 to 600 times more effectively than paraquat for reduction by three different flavo-containing enzymes; xanthine oxidase, NADH cytochrome c reductase, and NADPH cytochrome c reductase. Paraquat and methylene blue both interact with deflavo xanthine oxidase, indicating that neither electron acceptor reacted at the FAD site of the enzyme where molecular oxygen is reduced to superoxide. As the paraquat radical also directly reduced acetylated cytochrome c the hemeprotein could not be utilized for measuring superoxide production in the presence of the herbicide. In the presence of cytochrome c the methylene blue caused a sharp decrease in both paraquat-induced superoxide and hydroxyl radical production.
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PMID:Methylene blue competes with paraquat for reduction by flavo-enzymes resulting in decreased superoxide production in the presence of heme proteins. 283 6


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