Xanthine oxidase substrates

Boroxazothienopyridines, 4, 1029-1032 Borsche synthesis, 3, 44 Botrydiplodin mass spectrometry, 4, 585 Boulton-Katritzky rearrangement, 5, 288 Bovine milk xanthine oxidase substrates, 1, 234 Bradsher reaction... 

Hypoxanthine is oxidized at carbon 2 by both molybdenum hydroxylases, although xanthine oxidase is much more effective as a catalyst in this reaction [ 10]. A methyl substituent in this position prevents oxidation by either enzyme. Introduction of A-methyl substituents into the hypoxanthine nucleus produces dramatic effects on enzymic oxidation rates and also gives some insight into the productive modes of binding to each enzyme. Thus, it has been proposed that hypoxanthine tautomerizes in the xanthine oxidase-substrate complex to the 3-NH-form with a simultaneous shift of the NH-group in the imidazole ring from position 9 to 7 [ 198,200]. In support of this hypothesis, when tautomerism in the imidazole ring is prevented by substitution at N-7 or N-9, such compounds are almost refractory to oxidation (see Table 3.9)... 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Reperfusion of the synovial membrane occurs when exercise is stopped and O2 is subsequently reintroduced to the tissue. O2 is a substrate required for xanthine oxidase activity and O2" is generated. Therefore, repeated cycles of rest-exercise-rest in the inflamed joint may provide a continuous flux of destructive ROM. 

Exposure to xanthine oxidase plus Its substrates (xanthine, hypoxanthine)... 

Scheme 1. Possible oxidation-reduction reactions between reducing and oxidizing substrate molecules (R and O respectively) and the molybdenum (M), flavin (F) and iron (I) of xanthine oxidase. The enzyme molecule is represented by the circle and arrows indicate transfer of reducing equivalents...

When xanthine oxidase is treated anaerobically with xanthine at pH 8.2, the signals obtained depend in a complex way both on enzyme and substrate concentrations and on reaction time. Detailed examination of... 

Finally, in the case of inhibitory substrate analogues such as allo-xanthine, strong evidence has recently been presented that these bind to molybdenum in reduced xanthine oxidase (33). If the enzyme is reduced with xanthine, then treated anaerobically with alloxanthine and finally exposed to air, catalytic activity is lost. Though flavin and iron in the final product are in the oxidized state, there are significant spectral differences between it and the native enzyme. These are believed (33) due to reduction of molybdenum from Mo(VI) to Mo(IV) and complexing of... 

If existence of a persulphide or other potentially electron accepting sulphur group is confirmed, this might explain why redox titration experiments have shown the number of electron equivalents which the xanthine oxidase molecule can accept to be greater than is required for reduction of the three non-protein components (58, 91). Certainly, this interpretation seems more probable than the original suggestion (58, 91) that the molybdenum can be reduced to lower oxidation states than Mo(IV) by some substrates. 

A current overall picture of the reaction mechanism of xanthine oxidase, which differs substantially from one proposed earlier (87) is as follows. The enzyme is presumed to have two independent catalytic units, though this has not so far been proved rigorously. Reducing substrates are bound at molybdenum and reduce this from Mo(VI) both to Mo(V) and to Mo (IV). Reducing equivalents are then transferred by intramolecular reactions from molybdenum to iron-sulphur and also, either directly or via this, to flavin. Oxidizing substrates as a class, seem capable of reacting with all three types of centre in the enzyme. Thus, oxygen reacts predominantly with flavin, phenazine methosulphate... 

Xanthine dehydrogenase from chicken liver reacts readily with NAD as acceptor (77) while that from Micrococcus lactilyticus is inactive towards this, reacting instead with ferredoxin (18). Both enzymes react only slowly with oxygen. It seems reasonable to assume, however, that for each member of this group of enzymes, reducing substrates all react via molybdenum, as in milk xanthine oxidase. Presumably, different... 

So little is known about molybdenum enzymes other than milk xanthine oxidase that there is little to be said by way of general conclusions. In all cases where there is direct evidence (except possibly for xanthine dehydrogenase from Micrococcus lactilyticus) it seems that molybdenum in the enzymes does have a redox function in catalysis. For the xanthine oxidases and dehydrogenases and for aldehyde oxidase, the metal is concerned in interaction of the enzymes with reducing substrates. However, for nitrate reductase it is apparently in interaction with the oxidizing substrate that the metal is involved. In nitrogenase the role of molybdenum is still quite uncertain. 

Most in vitro studies of xanthines have centered around the enzyme xanthine oxidase. Bergmann and co-workers 40-4)) have examined the main oxidative pathways in the xanthine oxidase catalyzed oxidation of purines. The mechanism proposed by these workers 41 > is that the enzyme binds a specific tautomeric form of the substrate, regardless of whether or not that form represents the major structure present in solution. It is then proposed that the purine, e.g., xanthine, undergoes hydration at the N7=C8 double bond either prior to or simultaneously with dehydrogenation of the same position. Accordingly, the process would involve either pathway a or b. Fig. 15. Route a would give a lactim form of the oxidized purine, while b would give the cor-... 

Nitric oxide is a physiological substrate for mammalian peroxidases [myeloperoxide (MPO), eosinophil peroxide, and lactoperoxide), which catalytically consume NO in the presence of hydrogen peroxide [60], On the other hand, NO does not affect the activity of xanthine oxidase while peroxynitrite inhibits it [61]. Nitric oxide suppresses the inactivation of CuZnSOD and NO synthase supposedly via the reaction with hydroxyl radicals [62,63]. On the other hand, SOD is able to modulate the nitrosation reactions of nitric oxide [64]. 

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