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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 pH dependence and solvent isotope sensitivity of three discrete steps in the reductive half-reaction of xanthine oxidase have been investigated. The pH dependence of both kcat/Km from steady-state experiments and kred/Kdfrom rapid reaction experiments with xanthine as substrate indicate that enzyme reacts preferentially with the neutral form of substrate and that an ionizable group in the active site having a pKa of approximately 6.6 must be unprotonated for reaction to take place. The solvent kinetic isotope effect on kred/Kd is 2.4, once a uniform shift on going to D2O of approximately 1 unit for both pKa values is taken into account. The pH dependence of the formation and decay of Ered-P formed in the course the reaction of xanthine oxidase with lumazine has also been examined. Formation of this complex exhibits bell-shaped pH dependence, with pKa values of 6.5 and 7.8, consistent with the results obtained with xanthine. Decay of the Ered-P complex is base-catalyzed with a pKa > 11 and exhibits a small solvent kinetic isotope effect of 1.7 at pH/D 8.5. By contrast, the catalytic intermediate giving rise to the "very rapid" EPR signal that is transiently observed in the course of the reaction of enzyme with the substrate 2-hydroxy-6-methylpurine is found to undergo acid-catalyzed breakdown with an associated pKa < 6. Formation and decay of this species exhibit solvent kinetic isotope effects of 2.0 and 3.5 at pH 10. The results are discussed in the context of a specific reaction mechanism for the reductive half-reaction of xanthine oxidase, in which discrete ionizations associated with the molybdenum center of the active site play critical roles in determining the magnitude of the rate constants by which the Mo(IV)-P and Mo(V)-P intermediates form and decay.
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PMID:The reductive half-reaction of xanthine oxidase. The involvement of prototropic equilibria in the course of the catalytic sequence. 863 99

The hydroxyl and some alkoxyl spin adducts of 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) are difficult to assign due to the remarkable similarity of their EPR spectra. The utility of resolving superhyperfine (SHF) structure followed by computer simulations has been demonstrated to assist in the assignment of EPR spectra with close values of hyperfine splitting constants, e.g., DMPO/ .OH and DMPO/.OR. Here, .OR is the alkoxyl radical derived from thermal decomposition of 2,2' -azobis (2-amidinopropane) hydrochloride (AAPH). In addition, two other spin traps, derivatives of 2H-imidazole 1-oxide, namely, 2,2,4-trimethyl-2H-imidazole 1-oxide (TMIO) and 2,2-dimethyl-4-phenyl-2H-imidazole 1-oxide (DMPIO), have been used in a model study to develop a procedure for distinguishing between oxygen-centered spin adducts. These results are compared with those for DMPO. TMIO and DMPIO spin traps provide more distinguishable individual spectra with .OH and AAPH-derived .OR radicals than the DMPO spin trap. The formation of DMPO/.OR(AAPH) and DMPIO/ .OR(AAPH) spin adducts was confirmed by mass spectrometry. The comparison of spin trapping by DMPO and 2H-imidazole 1-oxides using typical biological sources of other oxygen-centered radicals reveals application limits of these spin traps. For example, 2H-imidazole 1-oxides do not form superoxide spin adducts in the xanthine/xanthine oxidase system. Also, for the first time, experimental evidence is presented for SHF structure in spectra of TMIO and DMPIO spin adducts with .OH/.OD and .CH3/ .CD3 radical species.
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PMID:Oxygen-centered spin adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 2H-imidazole 1-oxides. 866 Dec 92

The specificity of 2,2,6,6-tetramethylpiperidine to singlet oxygen was shown using Rose Bengal as a singlet oxygen generator, and Xanthine-Xanthine Oxidase and KO2 as the sources for the superoxide radical. The highest concentration of produced-singlet oxygen occurred at 25% of O2 by Rose Bengal photosensitization. The linewidth of the EPR signal for photosensitized nitroxyl radical, increasing solvent polarity. Deuterated solvents enlarge the EPR signal intensity in a dose-dependent manner. No EPR signal increase was observed in xanthine-xanthine oxidase reaction or KO2 systems, indicating that TEMP does not react with the superoxide anion. Thus, reaction of TEMP with 1O2 is highly specific.
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PMID:The specificity and product of quenching singlet oxygen by 2,2,6,6-tetramethylpiperidine. 867 11

Acetylcholine-induced, endothelium-dependent relaxation of norepinephrine-precontracted aortic strips, was severely impaired after exposure to a hypoxanthine/xanthine oxidase reaction generating oxygen radicals. This effect was more evident in aortic strips of aging rats (24 months old) in comparison to young rats (3 months old). The addition of authentic .NO (1 microM) completely relaxed aortic strips exposed to oxidative stress both in young and aging rats. In vitro EPR measurements showed that the .NO signal was reduced by enzymatic O2.- generating reaction. The activity of a partial purified preparation of constitutive NO synthase from rat cerebellum was significantly decreased after exposure to exogenous oxygen radicals. Pretreatment of aortic strips with 100 microM alpha-tocopherol-phosphate, produced a significant improvement of acetylcholine-dependent relaxation in the aortic strips exposed to oxidative stress, particularly in the aged vessel. The content of malondialdehyde in aortic tissue did not change after oxidative stress or alpha-tocopherol pretreatment. Alpha-tocopherol was unable to recover the NO synthase activity depressed in vitro by hypoxanthine/xanthine oxidase reaction. This study confirms that an oxidative stress impairs the endothelium-mediated vasodilation. Alpha-tocopherol pretreatment protects the vessel against this damage. The mechanism of action of alpha-tocopherol is unknown, but seems unrelated to an antioxidant activity.
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PMID:Alpha-tocopherol pretreatment improves endothelium-dependent vasodilation in aortic strips of young and aging rats exposed to oxidative stress. 873 50

For three prokaryotic enzymes of the xanthine oxidase family, namely quinoline 2-oxidoreductase, quinaldine 4-oxidase, and isoquinoline 1-oxidoreductase, the electron transfer centers were investigated by electron paramagnetic resonance. The enzymes are containing a molybdenum-molybdopterin cytosine dinucleotide cofactor, two distinct [2Fe-2S] clusters and, apart from isoquinoline 1-oxidoreductase, a flavin adenine dinucleotide. The latter cofactor yields two different organic radical signals in quinoline 2-oxidoreductase and quinaldine 4-oxidase, typical for the neutral and anionic form, respectively. A "rapid" Mo(V) species is present in all enzymes with small differences in magnetic parameters. From spectra simulation of 95Mo-substituted quinoline 2-oxidoreductase, a deviation of 25 degrees between the maximal g and 95Mo-hyperfine tensor component was derived. The very rapid Mo(V) species was detected in small amounts upon reduction with substrates in quinoline 2-oxidoreductase and quinaldine 4-oxidase, but showed a different kinetic behavior with considerable EPR intensities in isoquinoline 1-oxidoreductase. The FeSI and FeSII centers produced different signals in all three enzymes and, in case of isoquinoline 1-oxidoreductase, revealed a dipolar interaction, from which a maximum distance of 15 A between FeSI and FeSII was estimated. The midpoint potentials of the FeS centers were surprisingly different and determined for FeSI/FeSII with -155/-195 mV in quinoline 2-oxidoreductase, -250/-70 mV in quinaldine 4-oxidase, and +65/+10 mV in isoquinoline 1-oxidoreductase. The slopes of the fitting curves for the Nernst equation are indicative for nonideal behavior. Only in quinoline 2-oxidoreductase, an averaged midpoint potential of the molybdenum redox pairs of about -390 mV could be determined. Both of the other enzymes did not produce Mo(V) signals in redox titration experiments, probably because of direct reduction of Mo(VI) to Mo(IV) in the presence of dithionite.
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PMID:Comparative EPR and redox studies of three prokaryotic enzymes of the xanthine oxidase family: quinoline 2-oxidoreductase, quinaldine 4-oxidase, and isoquinoline 1-oxidoreductase. 924 10

Tetrahydroisoquinolines (TIQs) are endogenous compounds deriving from the nonenzymatic Pictet-Spengler condensation of catecholamines (CA) with aldehydes. TIQs have been extensively studied in the last years not only because they have been found in the brain of postmortem specimens of Parkinson's patients, but also because they are able to induce parkinsonian symptoms if injected in animals. In the present article we demonstrate that TIQs bearing a catecholic moiety (tetrahydropapaveroline, salsolinol, laudanosoline, and apomorphine) are easily oxidized in the presence of hydrogen peroxide by various enzymes--i.e., peroxidase (POD), lipoxygenase (LOX), and xanthine oxidase (XO)--into the corresponding TIQ-melanins. The kinetic parameters of the above-mentioned reactions and some spectroscopic characteristics of the synthetized pigments are reported. In particular, UV-VIS and EPR spectra emerge as very similar to those exhibited by dopa-melanin. Furthermore, TIQ-melanins appear to be similar to dopa-melanin regarding some specific physico-chemical properties: NADH-oxidizing properties, oxy-radicals scavenging activity, and ability to form soluble mixed polymers with melanins from opioid peptides.
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PMID:Melanins from tetrahydroisoquinolines: spectroscopic characteristics, scavenging activity and redox transfer properties. 943 26

Treatment of xanthine oxidase with ferricenium at high pH gives rise to an EPR signal not previously seen with this enzyme. The signal is apparently isotropic at 9 GHz with a gavg of approximately 2 and once generated is stable to pH 6.0, so long as the sample is kept in the dark. Treatment of the signal-giving species with hydroxyurea results in complete loss of the signal, indicating that the signal is radical-based. Pretreatment of the enzyme with iodoacetate has no effect on signal formation with ferricenium. The ferricenium-generated EPR signal shows proton hyperfine coupling that is not lost upon exchange into D2O and bears considerable resemblance to the tyrosyl radical of the photosynthetic reaction center and other systems. These observations lead us to interpret the new ferricenium-generated EPR signal of xanthine oxidase as arising from a tyrosyl radical, the result of one-electron oxidation of a protein tyrosinate residue. Kinetic parameters for the reductive half-reaction of ferricenium-treated xanthine oxidase with xanthine were determined by stopped-flow spectrophotometry; kred and KDxanthine (15 s-1 and 12 microM, respectively) were essentially unchanged. Addition of 2-hydroxy-6-methylpurine (in the presence of 2 mg/mL catalase and superoxide dismutase) generated the "very rapid" MoV EPR signal while preserving the ferricenium-derived EPR signal, providing a further indication that the modified enzyme remains fully functional and the presence of the tyrosyl radical does not impact turnover by the enzyme. Coupling of the two signals was not evident, nor was coupling to the two 2Fe-2S centers or the flavin semiquinone evident. The implications of covalent modifications of proteins mediated by ferricenium are discussed.
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PMID:Formation of a tyrosyl radical in xanthine oxidase. 960 Oct 39

The direct effects of the neurohormone melatonin on reactive oxygen species (ROS) were investigated. Melatonin was found to inhibit DMPO-O-2 formation in a dose-dependent manner. At the level of 1. 7+/-0.07 mM, melatonin caused 50% inhibition of EPR signal intensity of DMPO-O-2 during the reaction of xanthine and xanthine oxidase. The reaction rate constant of melatonin with O2- was found to be 1.25+/-0.07x103 M-1 s-1. However, melatonin (up to 1.2 mM) did not exhibit significant effect toward OH radical, produced by the Fenton reaction. In addition, we found no evidence for the formation of the melatonin indolyl cation radical that presumably precedes conversion of melatonin to its stable N1-acetyl-N2-5-methoxykynuramine (AMK) metabolite following sequential reactions of melatonin with O2- and OH. On the other hand, melatonin was capable of scavenging H2O2 in a dose-dependent manner with an IC50=0.5+/-0.02 mM. The reaction rate constant of melatonin with H2O2 was found to be 2.52+/-0.19x105 M-1 s-1. Furthermore, melatonin was also found to inhibit 1O2-dependent 2,2,6,6-tetramethylpiperidine oxide (TEMPO) radical formation during rose bengal photodynamic reaction. The results suggest that melatonin's antioxidant properties, in part, may involve a direct effect on scavenging of ROS.
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PMID:Scavenging of reactive oxygen species by melatonin. 983 10

In order to investigate the effects of trace elements on different metabolic pathways, the thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius (DSM 639) has been cultivated on various carbon substrates in the presence and absence of molybdate. When grown on glucose (but neither on glutamate nor casein hydrolysate) as sole carbon source, the lack of molybdate results in serious growth inhibition. By analysing cytosolic fractions of glucose adapted cells for molybdenum containing compounds, an aldehyde oxidoreductase was detected that is present in the cytosol to at least 0.4% of the soluble protein. With Cl2Ind (2,6-dichlorophenolindophenol) as artificial electron acceptor, the enzyme exhibits oxidizing activity towards glyceraldehyde, glyceraldehyde-3-phosphate, isobutyraldehyde, formaldehyde, acetaldehyde and propionaldehyde. At its pH-optimum (6.7), close to the intracellular pH of Sulfolobus, the glyceraldehyde-oxidizing activity is predominant. The protein has an apparent molecular mass of 177 kDa and consists of three subunits of 80.5 kDa (alpha), 32 kDa (beta) and 19.5 kDa (gamma). It contains close to one Mo, four Fe, four acid-labile sulphides and four phosphates per protein molecule. Methanol extraction revealed the existence of 1 FAD per molecule and 1 molybdopterin per molecule, which was identified as molybdopterin guanine dinucleotide on the basis of perchloric acid cleavage and thin layer chromatography. EPR-spectra of the aerobically prepared enzyme exhibit the so-called 'desulpho-inhibited'-signal, known from chemically modified forms of molybdenum containing proteins. Anaerobically prepared samples show both, the signals arising from the active molybdenum-cofactor as well as from the two [2Fe-2S]-clusters. According to metal-, cofactor-, and subunit-composition, the enzyme resembles the members of the xanthine oxidase family. Nevertheless, the melting point and long-term thermostability of the protein are outstanding and perfectly in tune with the growth temperature of S. acidocaldarius (80 degrees C). The findings suggest the enzyme to function as a glyceraldehyde oxidoreductase in the course of the nonphosphorylated Entner-Doudoroff pathway and thereby may attribute a new physiological role to this class of enzyme.
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PMID:The strict molybdate-dependence of glucose-degradation by the thermoacidophile Sulfolobus acidocaldarius reveals the first crenarchaeotic molybdenum containing enzyme--an aldehyde oxidoreductase. 1009 93

The increasing knowledge on the participation of free radicals in many diverse clinical and pathological conditions, has consequently expanded the search for new and versatile antioxidants aimed at combating oxidative stress. Our interest in this field concerns aromatic indolinonic aminoxyls (nitroxides) which efficiently react with alkoxyl, peroxyl, aminyl, arylthiyl and alkyl radicals to give non-paramagnetic species. This prompted us to test their antioxidant activity on different biological systems exposed to free radical-induced oxidative stress and the results obtained so far have been very promising. However little is known about their behaviour towards superoxide and hydroxyl radicals. Here, we report on the reactivity of an indolinonic aminoxyl, with the two above mentioned radicals using hypoxanthine/xanthine oxidase and potassium superoxide for generating the former and the Fenton reagent for the latter. Besides performing the deoxyribose assay for studying the reaction of the aminoxyl with hydroxyl radical and monitoring spectral changes of the aminoxyl in the presence of superoxide radical, macroscale reactions were performed in both cases and the products of the reactions isolated and identified. The EPR technique was used in this study to help elucidate the data obtained. The results show that this compound efficiently reacts with both hydroxyl and superoxide radicals and furthermore, it is capable of maintaining iron ions in its oxidized form. The results thus contribute to increasing the knowledge on the reactivity of indolinonic aminoxyls towards free radical species and as a consequence, these compounds and/or other aminoxyl derivatives, may be considered as complementary, and sometimes alternative sources for combating oxidative damage.
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PMID:Reactivity of an indolinonic aminoxyl with superoxide anion and hydroxyl radicals. 1049 Feb 40


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