Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.17.3.2 (xanthine oxidase)
8,383 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The properties of the molybdenum iron-sulfur flavoprotein, aldehyde oxidase from rabbit livers, have been further investigated in comparison with bovine milk xanthine oxidase. In agreement with earlier work, the ultraviolet/visible spectra indicate that the flavin and iron-sulfur centres of the enzymes are quite similar to one another. The molybdenum centres have been compared by EPR spectroscopy of molybdenum(V) and regarding re-insertion of the sulfido ligand of molybdenum into the desulfo enzyme forms. The pH optimum for sulfide insertion is approximately 2 lower for aldehyde oxidase than for xanthine oxidase. A detailed comparison of molybdenum(V) EPR signals has been made for the signals known as Arsenite, Slow and Rapid. Computer simulation of spectra in 1H2O and 2H2O, at 9 and 35 GHz was used. Slow signals from the two enzymes are scarcely distinguishable from one another. Under the conditions used, aldehyde oxidase yielded only the Rapid type 2 signal, whereas xanthine oxidase gives both the Rapid type 1 and 2 signals. The nature of the structural difference between the Rapid type 1 and type 2 signal-giving species is discussed. It is concluded that the molybdenum centres of xanthine oxidase and aldehyde oxidase are indeed similar to one another and that such differences as exist between their molybdenum(V) EPR signals and re-sulfuration properties are related to differences only in the substrate-binding sites. N-terminal amino acid analyses have been performed on peptides obtained by trypsin cleavage of aldehyde oxidase. Comparison with a sequence previously deduced [Wright, R. M., Vaitaitis, G. M., Wilson, C. M., Repine, T. B., Terada, L. S. & Repine, J. E. (1993) Proc. Natl Acad. Sci. USA 90, 10690-10694] makes it clear that the latter is not, as was assumed, that of a xanthine dehydrogenase but of an aldehyde oxidase. In contrast to the situation with xanthine oxidase, attempts to convert non-proteolysed aldehyde oxidase to a dehydrogenase form by treatment with dithiothreitol were unsuccessful. The reason for this is considered in the light of sequence data in the literature. The location of the NAD(+)-binding site is discussed, and the sequence data are also discussed in relation to the molybdenum, iron-sulfur and substrate-binding sites.
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PMID:Properties of rabbit liver aldehyde oxidase and the relationship of the enzyme to xanthine oxidase and dehydrogenase. 755 19

Manganese-containing superoxide dismutases (Mn-SODs) and iron-containing superoxide dismutases (Fe-SODs) from aerobic bacteria often show high metal specificity for their enzymic activities by a standard assay system using xanthine-xanthine oxidase and cytochrome c. In this study, we have attempted to characterize the structural basis of the metal specificity of manganese-containing SOD (Mn-SOD) using Fe-substituted Mn-SOD prepared from apo-Mn-SOD from Serratia marcescens. The Fe3+ content of the Fe-substituted enzyme was 1.71 +/- 0.14 mol/mol dimer and the specific activity was 34.8 +/- 4.8 units.mg protein-1.mol Fe3+(-1).mol subunit-1. Fe-substituted Mn-SOD was found to react with the superoxide anion at pH 8.1 with a second-order rate constant of 6 x 10(6) M-1 s-1, which is approximately 1% of that of native Mn-SOD at the same pH. However, the rate constant increased with decreasing pH to approximately 10% (5 x 10(7) M-1 s-1) that of native Mn-SOD at pH 6.0 with a pK of 7.0. The visible absorption spectrum and EPR spectrum of Fe-substituted Mn-SOD also showed pH-dependent changes with pK values of 6.6 and 7.2, respectively. Similarly, the affinity of the azide ion, an analog of the superoxide ion, for iron of Fe-substituted Mn-SOD increased with decreasing pH, with a pK value of 7.0 (e.g. Kd = 0.1 mM at pH 6.2 and 0.9 mM at pH 8.2). The similarity of these pK values suggests that the activity, the spectral changes and the affinity of the azide ion for iron are derived from the same change in the metal environment. After comparison with the reported pK values (around 9) of similar pH-dependent changes in the spectra, the enzymic activity and the affinity of azide for iron of Fe-SOD from Escherichia coli, we proposed that the difference in the pK values of a hydroxide ion binding to iron between Fe-substituted Mn-SOD and Fe-SOD may cause the different pH dependencies of these changes in each SOD.
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PMID:The pH-dependent changes of the enzymic activity and spectroscopic properties of iron-substituted manganese superoxide dismutase. A study on the metal-specific activity of Mn-containing superoxide dismutase. 786 28

The dimethylsulphoxide reductase of Rhodobacter capsulatus contains a pterin molybdenum cofactor molecule as its only prosthetic group. Kinetic studies were consistent with re-oxidation of the enzyme being rate limiting in the turnover of dimethylsulphoxide in the presence of the benzyl viologen radical. EPR spectra of molybdenum(V) were generated by reducing the highly purified enzyme under a variety of conditions, and with careful control it was possible to generate at least five clearly distinct EPR signals. These could be simulated, indicating that each corresponds to a single chemical species. Structures of the signal-giving species are discussed in light of the EPR parameters and of information from the literature. Three of the signals show coupling of molybdenum to an exchangeable proton and, in the corresponding species, the metal is presumed to bear a hydroxyl ligand. One signal with gav 1.96 shows a very strong similarity to a signal for the desulpho form of xanthine oxidase, while two others with gav values of 1.98 show a distinct similarity to signals from nitrate reductase of Escherichia coli. These data indicate an unusual flexibility in the active site of dimethylsulphoxide reductase, as well as emphasising structural similarities between molybdenum enzymes bearing different forms of the pterin cofactor. Interchange among the different species must involve either a change of coordination geometry, a ligand exchange, or both. The latter may involve replacement of an amino acid residue co-ordinating molybdenum via O or N, for a cysteine co-ordinating via S. Since the two signals with gav 1.96 were obtained only under specific conditions of reduction of the enzyme by dithionite, it is postulated that their generation may be triggered by reduction of the pteridine of the molybdenum cofactor from a dihydro state to the tetrahydro state.
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PMID:Multiple states of the molybdenum centre of dimethylsulphoxide reductase from Rhodobacter capsulatus revealed by EPR spectroscopy. 792 52

A spin-labeled adenine derivative [N6-(2,2,6,6-tetramethyl-1-oxypiperidin-4-yl)adenine; SLAD] is found to be a very slow substrate of xanthine oxidase based on the observed reduction of enzyme by SLAD under anaerobic conditions. A room-temperature EPR spectrum of SLAD in the presence of oxidized xanthine oxidase shows the appearance of "wings" on the three-line spectrum of the free spin-label, indicating formation of an E.SLAD complex. This spectrum can be obtained on a timescale that is short compared to catalysis. Using this spectral change as an experimental probe, the room-temperature Kd's of SLAD binding to oxidized xanthine oxidase at various pH's have been determined. Obtained Kd values are 1.5 +/- 0.3 mM, 1.6 +/- 0.3 mM, and 1.5 +/- 0.3 mM at pH 10.0, 8.5, and 7.0, respectively, indicating no significant difference in the equilibrium dissociation of SLAD from enzyme upon pH change. These results are consistent with the calculated equilibrium dissociation constant for substrate binding to oxidized molybdenum center based on Kd to reduced enzyme and the perturbation of MoVI/MoV and MoV/MoIV reduction potentials by product and substrate analogs.
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PMID:Studies of the substrate binding to xanthine oxidase using a spin-labeled analog. 796 16

Iodonium inhibition of the flavoenzymes neutrophil NADPH oxidase and cytochrome P450 reductase has been suggested to require reductive metabolism of the inhibitor to a phenyl radical. Inhibition would ultimately result from covalent attachment of phenyl radicals to either the flavin cofactor or adjacent amino acid side chains important in catalysis. In this paper we provide evidence, using EPR techniques, that phenyl radicals are formed during reaction of iodonium diphenyl with reduced free flavin (FMN) and protein-bound (cytochrome P450 reductase or xanthine oxidase) flavin. Kinetic analysis indicated iodonium diphenyl to be an uncompetitive inhibitor of xanthine oxidase, suggesting the need for reduced enzyme for inhibition. A study of the catalytic and structural properties of different flavoenzymes suggested that only enzymes containing flavins that function in one-electron transfer are targets for iodonium inhibition.
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PMID:Involvement of phenyl radicals in iodonium inhibition of flavoenzymes. 796 60

A method was developed to measure the superoxide generation rate from biological systems using the spin trapping method. Kinetic treatment of the decay rate of the superoxide adduct of 5,5-dimethylpyrroline N-oxide (DMPO) revealed that the EPR signal intensity of the system is proportional to the superoxide generation rate. Rapid depletion of oxygen in the sample was utilized to terminate superoxide generation so that the decay rate of the DMPO superoxide adduct (DMPO-OOH) could be determined. For this decay measurement, a controlled atmosphere EPR cavity was developed and was used with an open-air sample cell. Superoxide generation rates determined with this method for stimulated neutrophils and for the hypoxanthine-xanthine oxidase system were comparable to those obtained with the cytochrome c reduction method. This method is specifically applicable to the system in which dissolved oxygen supplied from the gas phase is utilized as a source of superoxide.
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PMID:Determination of the rate of superoxide generation from biological systems by spin trapping: use of rapid oxygen depletion to measure the decay rate of spin adducts. 798 27

The pulsed EPR technique of electron spin echo envelope modulation (ESEEM) has been utilized to examined both the 'very rapid' and 'desulfo inhibited' Mo(V) signals of xanthine oxidase in order to probe for magnetic interactions with nitrogen, phosphorus and hydrogen nuclei. No 14N modulation is observed in the 'desulfo inhibited' EPR signal, indicating that histidine is unlikely to be a ligand to molybdenum. Strong 14N modulation is observed in the 'very rapid' EPR signal formed with 2-hydroxy-6-methylpurine substrate bound to molybdenum. We interpret this modulation as arising from nitrogens of the bound purine substrate. This interpretation is consistent with the present evidence indicating that the purine ring present in the species giving rise to the 'very rapid' EPR signal is coordinated to the molybdenum center through the catalytically introduced hydroxyl group. No modulation is observed from non-exchangeable deuterons in experiments performed with deuterated 2-hydroxy-6-methylpurine. Given the signal-to-noise level of the spectra, the lack of modulation indicates that each of the substrate methyl group deuterons is greater than 4.9 A from the Mo(V). The deuteron removed from the C8 position in the binding of the substrate is also exchanged to a site or sites greater than 4.9 A from the Mo(V) in the time-course of sample preparation. Moderately deep deuteron modulation arises from exchangeable sites. A large portion of this modulation can be accounted for by the exchangeable N7 deuteron of the 2-hydroxy-6-methylpurine substrate, which we estimate to be approximately 3.2 A from the molybdenum. Additional exchangeable deuterons on the protein or within the buffer must be present within 5 A of the molybdenum to account for the remaining modulation. No modulation from weakly-coupled 31P nuclei is observed in either the 'desulfo inhibited' or 'very rapid' EPR signal.
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PMID:Electron spin echo envelope modulation spectroscopy of the molybdenum center of xanthine oxidase. 818 Feb 33

Combined stimulation, by superoxide ions generated by the xanthine-xanthine oxidase reaction, and platelet-activating factor (PAF), induced cell differentiation of rat monocytic leukemia cells (c-WRT-LR) to macrophage-like mature cells. Monitoring of cytochrome c reduction revealed that PAF stimulation induced the release of superoxide ions from c-WRT-LR. To further investigate the effect of superoxide ions in the autocrine or paracrine mechanism in cell differentiation, molecular species of the oxygen radicals under PAF stimulation were examined using the EPR spin trap, 5,5'-dimethyl-1-pyrroline N-oxide (DMPO). PAF and/or phorbol myristate acetate caused the formation of EPR spectra, a combination of DMPO/.OOH and DMPO/.OH. Since both spectra were diminished in the presence of superoxide dismutase, it was concluded that DMPO/.OH was derived from superoxide ions. Mannitol and catalase suppressed cell differentiation induced by combined stimulation with PAF and oxygen radicals generated by the xanthine-xanthine oxidase reaction. Taken together, these results suggest that hydroxyl radicals generated by Fenton reaction from H2O2 may be involved in the mechanism of cell differentiation in rat monocytic leukemia cells.
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PMID:A role for oxygen radicals in rat monocytic leukemia cell differentiation under stimulation with platelet-activating factor. 830 1

The pH dependence of the behavior of chicken liver xanthine dehydrogenase in the course of reductive titrations with sodium dithionite has been examined. Below pH 8.5, the behavior of xanthine dehydrogenase is similar to that of the much better understood milk xanthine oxidase, with the amount of neutral semiquinone accumulating transiently in the course of the titration increasing somewhat as the pH decreases. At pH 10, however, an anomalously large accumulation of the neutral semiquinone is observed by both UV/visible and EPR spectroscopy. Treatment of xanthine dehydrogenase with the thiol reagent iodoacetamide significantly diminishes the ability of the enzyme to stabilize the neutral flavin semiquinone at high pH. These data are consistent with the presence of a protein thiol in the immediate vicinity of the flavin, whose ionization above pH 8.5 results in thermodynamic stabilization of the neutral flavin semiquinone over the anionic form.
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PMID:Paradoxical stabilization of the neutral flavin semiquinone of xanthine dehydrogenase at high pH. 839

The reaction mechanism of the molybdoenzyme xanthine oxidase has been further investigated by 13C and 17O ENDOR of molybdenum(V) species and by kinetic studies of exchange of oxygen isotopes. Three EPR signal-giving species were studied: (i) Very Rapid, a transient intermediate in substrate turnover, (ii) Inhibited, the product of an inhibitory side reaction with aldehyde substrates, and (iii) Alloxanthine, a species formed by reaction of reduced enzyme with the inhibitor, alloxanthine. The Very Rapid signal was developed either with [8-13C]xanthine or with 2-oxo-6-methylpurine using enzyme equilibrated with [17O]H2O. The Inhibited signal was developed with 2H13C2HO and the Alloxanthine signal by using [17O]H2O. Estimates of Mo-C distances were made, from the anisotropic components of the 13C-couplings, by corrected dipolar coupling calculations and by back-calculation from assumed possible structures. Estimated distances in the Inhibited and Very Rapid species were about 1.9 and less than 2.4 A, respectively. A Mo-C bond in the Inhibited species is very strongly suggested, presumably associated with side-on bonding to molybdenum of the carbonyl of the aldehyde substrate. For the Very Rapid species, a Mo-C bond is highly likely. Coupling from a strongly coupled 17O, not in the form of an oxo group, and no coupling from other oxygens was detected in the Very Rapid species. No coupled oxygens were detected in the Alloxanthine species. That the coupled oxygen of the Very Rapid species is the one that appears in the product uric acid molecule was confirmed by new kinetic data. It is concluded that this oxygen of the Very Rapid species does not, as frequently assumed, originate from the oxo group of the oxidized enzyme. A new turnover mechanism is proposed, not involving direct participation of the oxo ligand group, and based on that of Coucouvanis et al. [Coucouvanis, D., Toupadakis, A., Lane, J. D., Koo, S. M., Kim, C. G., Hadjikyriacou, A. (1991) J. Am. Chem. Soc. 113, 5271-5282]. It involves formal addition of the elements of the substrate (e.g., xanthine) across the Mo = S double bond, to give a Mo(VI) species. This is followed by attack of a "buried" water molecule (in the vicinity of molybdenum and perhaps a ligand of it) on the bound substrate carbon, to give an intermediate that on intramolecular one-electron oxidation gives the Very Rapid species. The latter, in keeping with the 13C, 17O, and 33S couplings, is presumed to have the 8-CO group of the uric acid product molecule bonded side-on to molybdenum, with the sulfido molybdenum ligand retained, as in the oxidized enzyme.
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PMID:Evidence favoring molybdenum-carbon bond formation in xanthine oxidase action: 17Q- and 13C-ENDOR and kinetic studies. 863 73


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