Mechanism xanthine oxidation

There are probably more publications relating to xanthine oxidase than to any other enzyme studied, certainly more than those pertaining to aldehyde oxidase. This is presumably because the former enzyme is easily accessible from cow s milk rather than from animal tissue. It is not the purpose of this review to include all the data amassed on xanthine oxidase, as this has been fully covered in recent reviews [8, 12, 13]. Furthermore, most of our own work has been concerned with aldehyde oxidase. Thus, this report compares the properties of the molybdenum hydroxylases, where possible, in terms of distribution, substrate and inhibitor specificity and mechanism of oxidation. 

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... 

Fig. 8. Primary electrochemical mechanism and products formed on oxidation of xanthine at the PGE in 1 M HOAc...

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-... 

In the case of the methylated xanthines, particularly theophylline, theobromine and caffeine, the preponderance of data on the metabolism of these compounds in man suggests that a methylated uric acid is the principal product. However, the data presented earlier proposes at best a 77 per cent accounting of the methylated xanthine administered. The question can be raised as to whether the final products observed upon electrochemical oxidation of these compounds aids these studies. Very recently studies of metabolism of caffeine have revealed that 3,6,8-trimethylallantoin is a metabolite of caffeine 48>. This methylated allantoin is, of course, a major product observed electrochemically. The mechanism developed for the electrochemical oxidation seems to nicely rationalize the observed products and electrochemical behavior. The mechanism of biological oxidation could well be very similar, although insufficient work has yet been performed to come to any definite conclusions. There is however, one major difference between the electrochemical and biological reactions which is concerned with the fact that in the former situation no demethylation occurs whereas in the latter systems considerable demethylation appears to take place. 

The initial electrochemical and biological oxidation with xanthine oxidase are essentially identical. However, electrochemically 2,8-dioxyadenine the final product in the presence of xanthine oxidase is much more readily oxidizable than adenine 59) so that considerable further oxidation occurs. To the authors knowledge, 2,8-dioxyadenine is not a major metabolite of adenine in man or other higher organisms. Accordingly, it is likely that other enzymes accomplish further degradation of 2,8-dioxyadenine. The relationship between the products so formed and the mechanism of the reaction to the related electrochemical processes has yet to be studied. 

FIGURE 6.11 Typical current—time responses of Fe-SOD/MPA-modified Au electrode toward 02 in 25 mM phosphate buffer (02-saturated, pH 7.5) containing 0.002 unit of XOD upon the addition of 50 nM xanthine at +300 (a) and —lOOmV (b). The arrows represent the addition of 10 j,M of Cu, Zn-SOD (a) and 580 units of catalase and 10 pM of Cu, Zn-SOD to the solution (b). The solution was stirred with a magnetic stirrer at 200rpm. Inset mechanism for the amperometric response of SODs/MPA-modified Au electrodes to 02, based on enzymatic catalytic oxidation (a) and reduction (b) of 02 (M metal ions of SODs). (Reprinted from [138], with permission from the American Chemical Society.)... 

The release of iron from ferritin can be induced by different factors. In 1984, Biemond et al. [159] have shown that stimulated leukocytes mobilize iron from human and horse ferritin. Release of iron was induced by superoxide because SOD inhibited this process. Similarly, the release of iron from ferritin can be induced by xanthine oxidase [160] this process is believed to induce ischemia and inflammation. Under anerobic conditions xanthine oxidase is also able to stimulate iron release from ferritin through superoxide-independent mechanism [161]. Another physiological free radical nitric oxide also stimulates iron release from ferritin [162],... 

In 1974, Olson et al. [9] proposed a mechanism for the reactions catalyzed by XO. In accord with this mechanism six electrons are transferred from fully reduced enzyme through four redox centers during the oxidation of xanthine (Reaction (1)) ... 

The mechanism of iron-initiated superoxide-dependent lipid peroxidation has been extensively studied by Aust and his coworkers [15-18]. It was found that superoxide produced by xanthine oxidase initiated lipid peroxidation, but this reaction was not inhibited by hydroxyl radical scavengers and, therefore the formation of hydroxyl radicals was unimportant. Lipid peroxidation depended on the Fe3+/Fe2+ ratio, with 50 50 as the optimal value [19]. Superoxide supposedly stimulated peroxidation both by reducing ferric ions and oxidizing ferrous ions. As superoxide is able to release iron from ferritin, superoxide-promoted lipid peroxidation can probably proceed under in vivo conditions [16,20]. 

Inhibition and stimulation of LOX activity occurs as a rule by a free radical mechanism. Riendeau et al. [8] showed that hydroperoxide activation of 5-LOX is product-specific and can be stimulated by 5-HPETE and hydrogen peroxide. NADPH, FAD, Fe2+ ions, and Fe3+(EDTA) complex markedly increased the formation of oxidized products while NADH and 5-HETE were inhibitory. Jones et al. [9] also demonstrated that another hydroperoxide 13(5)-hydroperoxy-9,ll( , Z)-octadecadienoic acid (13-HPOD) (formed by the oxidation of linoleic acid by soybean LOX) activated the inactive ferrous form of the enzyme. These authors suggested that 13-HPOD attached to LOX and affected its activation through the formation of a protein radical. Werz et al. [10] showed that reactive oxygen species produced by xanthine oxidase, granulocytes, or mitochondria activated 5-LOX in the Epstein Barr virus-transformed B-lymphocytes. 

Thus, the mechanism of MT antioxidant activity might be connected with the possible antioxidant effect of zinc. Zinc is a nontransition metal and therefore, its participation in redox processes is not really expected. The simplest mechanism of zinc antioxidant activity is the competition with transition metal ions capable of initiating free radical-mediated processes. For example, it has recently been shown [342] that zinc inhibited copper- and iron-initiated liposomal peroxidation but had no effect on peroxidative processes initiated by free radicals and peroxynitrite. These findings contradict the earlier results obtained by Coassin et al. [343] who found no inhibitory effects of zinc on microsomal lipid peroxidation in contrast to the inhibitory effects of manganese and cobalt. Yeomans et al. [344] showed that the zinc-histidine complex is able to inhibit copper-induced LDL oxidation, but the antioxidant effect of this complex obviously depended on histidine and not zinc because zinc sulfate was ineffective. We proposed another mode of possible antioxidant effect of zinc [345], It has been found that Zn and Mg aspartates inhibited oxygen radical production by xanthine oxidase, NADPH oxidase, and human blood leukocytes. The antioxidant effect of these salts supposedly was a consequence of the acceleration of spontaneous superoxide dismutation due to increasing medium acidity. 

In addition to aconitases, nitric oxide is an inhibitor of many other enzymes such as ribonucleotide reductase [71], glutathione peroxidase [72,73], cytochrome c oxidase [74], NADPH oxidase [75], xanthine oxidase [76], and lipoxygenase [77] but not prostaglandin synthase [78]. (Mechanism of lipoxygenase inhibition by nitric oxide is considered in Chapter... 

It is usually believed that NO inhibits enzymes by reacting with heme or nonheme iron or copper or via the S-nitrosilation or oxidation of sulfhydryl groups, although precise mechanisms are not always evident. By the use of ESR spectroscopy, Ichimori et al. [76] has showed that NO reacts with the sulfur atom coordinated to the xanthine oxidase molybdenum center, converting xanthine oxidase into a desulfo-type enzyme. Similarly, Sommer et al. [79] proposed that nitric oxide and superoxide inhibited calcineurin, one of the major serine and threonine phosphatases, by oxidation of metal ions or thiols. 

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