Xanthine oxidase substrate binding

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)... 

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... 

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-... 

Xanthine oxidase also has a site that will bind anions such as nitrate. This appears to be the substrate-binding site on molybdenum. 

Over the course of study of xanthine oxidase, proposals for the mechanism by which the metal functions have ranged from hydride transfer to oxo transfer to CEPT linked to water activation [226,233-235], The latter proposal is presently favored, given what is known about (1) the source of the oxygen atom incorporated into product, (2) the fate of the substrate C(8)—H bond, (3) the role of the sulfido ligand, and (4) substrate binding. 

The decrease in rate of reaction of xanthine oxidase with the size of purine substrate is also consistent with a size-selective active site pocket and possible metal binding of substrate [242,243], A strongly coupled nitrogen is not observed in the very rapid signal, which is thought to include bound product, so it would appear that the urate is not N bound to the molybdenum center [152-158],... 

Possible Xanthine Oxidase Mechanism. The proposed reaction mechanism, (Figure 27), which must still be regarded as a working hypothesis, entails metal binding of substrate, metal-assisted activation of water, CEPT, and stabilization of the hydrosulfido ligand. 

Two of the carbonyl groups have gone and the imidazole ring has been replaced by a pyrazoie ring. Purines from DNA are degraded in the body to xanthine, which is oxidized to uric acid. Allopurinol binds to the enzyme xanthine oxidase but inactivates it by not reacting. In fact it imitates not uric acid but the true substrate xanthine in a competitive fashion. This enzyme plays a minor part in human metabolism so inhibiting it is not serious—it just prevents overproduction of uric acid. 

Kim, J. H., and Hille, R., 1994, Studies of substrate binding to xanthine oxidase by using a spin-labeled analog, J. Inorg. Biochem. 55 2959303. 

E4. Elion, G. B., Taylor, T. J., and Hitchings, G. H., Binding of substrates and inhibitor and xanthine oxidase from different species. Proc. 6th Intern. Congr. Biochem., New York, 1964 Vol. IV, p. 42. 

Administration of allopurinol, an analog of hypoxanthine, is one treatment for gout. The mechanism of action of allopurinol is interesting it acts first as a substrate and then as an inhibitor of xanthine oxidase. The oxidase hydroxylates allopurinol to alloxanthine (oxipurinol), which then remains tightly bound to the active site. The binding of alloxanthine keeps the molybdenum atom of xanthine oxidase in the + 4 oxidation state instead of it returning to the + 6 oxidation state as in a normal catalytic cycle. We see here another example of suicide inhibition. 

Because the molecular properties of aldehyde oxidase and xanthine oxidase do not differ significantly and they contain basically similar substrate-binding sites, it is often assumed that any mechanistic conclusions regarding one enzyme, usually the latter, can be also applied to the other [14, 51]. This may not be a valid assumption to make subtle alterations in the Mo centre may explain varying substrate specificities, but more fundamental modifications of the enzyme molecule may need to be considered to account for the differences in the position of substrate oxidation and, in particular, the rate-determining step. The oxidation of xanthine (3) or lumazine (4) catalysed by xanthine oxidase can be formulated as ... 

In this section are described the important chemical features of those substrates which are oxidized by the molybdenum hydroxylases. Although these enzymes, particularly aldehyde oxidase, also catalyse numerous reductive reactions under anaerobic conditions in vitro, it has not yet been established whether they occur under physiological conditions and there are as yet insufficient examples of any one reduction reaction to permit any conclusions regarding the structure of substrates. Thus, such reactions will not be discussed here (see [11] and references therein). Properties of those inhibitors which bind at the Mo centre and are also substrate analogues will also be included. However, the interaction of inhibitors such as cyanide and arsenite with the molybdenum hydroxylases and the mechanism of action of the specific xanthine oxidase inhibitor, allo-purinol, have been comprehensively described elsewhere [8, 12, 14, 157]. 

Although aldehyde oxidase and xanthine oxidase differ markedly in their substrate and inhibitor specificities, there is considerable evidence to suggest that binding of both substrates and inhibitors to either enzyme is facilitated by hydrophobic interaction in the enzyme active site. 

Simple unsubstituted monocycles such as pyridine, pyrazine, pyrimidine, pyridazine and pyrrole show little or negligible binding to either enzyme [3, 10, 173], However, we have shown that fusion or substitution of a phenyl group significantly increases the affinity of the compounds towards aldehyde oxidase, and in some cases the heterocycles are substrates for xanthine oxidase (Table 3.6) [50, 173]. Furthermore, studies with compounds containing a... 

Inhibition studies using xanthine oxidase also suggest that cationic substrates are oxidized at the same enzymic site as xanthine [ 179]. In this case, however, initial dissociation of an essential amino acid within the active site as a prerequisite is indicated before quaternary compounds bind, as these substrates are only very slowly oxidized at pH 7 and investigations have to be performed at pH > 9.6 [58, 179, 180]. Nevertheless, analogous substituent effects are observed with both molybdenum hydroxylases Table 3.7) and a relatively large hydrophobic binding site is again indicated with xanthine oxidase. 

Both molybdenum hydroxylases can oxidize uncharged and cationic substrates, although at physiological pH aldehyde oxidase is much more active towards the latter group of compounds. It does not necessarily follow, however, that when compounds have a pKa value around 7, the neutral and protonated forms both bind to the enzyme. Kinetic studies at different pH values have indicated that it is the unprotonated form of 2-aminophthalazine that reacts with the molybdenum hydroxylases, either as a substrate for aldehyde oxidase or a competitive inhibitor of xanthine oxidase [211]. In contrast, at pH 7-8 many of the purine and pteridine substrates are present as mixtures of uncharged molecules and monoanions, both of which are rapidly oxidized by xanthine oxidase [181, 200, 206, 210, 212]. 

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