Xanthine oxidase substrate specificities

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-... 

R is an electron-donor substrate such as purine or xanthine and A is an electron acceptor such as 02 or NAD+. It is thought that the in vivo mammalian form of xanthine oxidase uses NAD+ as acceptor and is therefore, more appropriately named xanthine dehydrogenase. No evidence exists for a dehydrogenase form of aldehyde oxidase. The specificities of xanthine oxidase and aldehyde oxidase have been extensively catalogued (96), and the mechanism and properties of these enzymes have been reviewed (97, 98). 

Let us for a moment consider the enzymes responsible for oxidation, the oxidases. Some are very specific as to the type of substrate which they will oxidize others are relatively nonspecific, such as the mixed function oxidases while some have a limited functional specificity such as monoamine oxidase, diamine oxidase and xanthine oxidase. Notice that the specific name for each enzyme will related in some way to its substrate. 

Aldehyde oxidase catalyzes the oxidation of aldehydes to carboxylic acids by dioxygen, but also catalyzes the hydroxylation of pyrimidines. Despite its rather broad specificity for substrates, it may well be that aldehyde oxidase should be regarded primarily as a pyrimidine hydroxylase. Thus, xanthine oxidase and aldehyde oxidase catalyze the hydroxylation of purines and pyrimidines respectively. The oxygen incorporated into the product comes from water, not 02. The dioxygen serves as the electron acceptor and other oxidizing agents may be used. 

A number of other enzymes, such as monoamine oxidase, alcohol dehydrogenase and xanthine oxidase, are also involved in drug metabolism. These enzymes tend to be more specific, oxidizing xenobiotics related to the normal substrate for the enzyme. 

The urinary caffeine test is not based on assays of specific substrates and products of NAT2 ( including other metabolism pathways involving at least xanthine-oxidases), and is affected by diet habits, xanthine-oxidase inhibitors such as allopurinol (Fuchs 1999), or other drugs (Klebovitch 1995). NAT activities are affected by anti-inflammatory drugs. Of note, acetominophen is an inhibitor of NAT2 in vivo (Rothen 1998). 

There are a number of molybdenum-containing enzymes, but those that are important in carbon oxidation of xenobiotics are aldehyde oxidase (AO) and xanthine oxidase (XO), also referred to as molybdenum hydroxylases (Figure 10.7). Both enzymes catalyze the oxidation of a wide range of aldehydes and N-heterocycles. The name aldehyde oxidase is somewhat misleading, however, because oxidation of heteroaromatics is more significant. The differences in substrate specificities between... 

Xanthine oxidase and related hydroxylase enzymes exhibit broad substrate specificity and an apparently complex catalytic cycle (109, 237). The important centers identified by EXAFS and EPR studies have... 

Activation of drugs to give toxic products is common. Apart from non-enzymatic activation (e.g., via autoxidation), activation by enzymatic one-electron oxidation or reduction frequently occurs. Several non-specific oxidases and reductases are encountered in mammalian tissues. Enzyme systems that have been studied in detail are peroxidases and microsomal oxidases and reductases. Xanthine oxidase also has received some attention. In many insta .ces the end products of the reaction are critically dependent upon the presence of oxygen in the system. This is because oxygen is an excellent electron acceptor, i.e., it can oxidize donor radicals, forming superoxide in the process. In this way a redox cycle is set up in which the xenobiotic substrate is recovered. The toxic effects of the xenobiotic often can be attributed to the oxidative stress arising from such a cycle. However, it seems that for some substrates, oxidative stress of this kind can be less damaging than anaerobic reduction. Anaerobic reduction can lead to formation of further reduced products with additional toxicity. 

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. 

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]. 

The oxidation of substituted benzaldehydes by xanthine oxidase is sterically hindered by bulky substituents at the ortho (o) position (Table 3.5) [167], Increasing the size of the halo-substituent dramatically decreases the oxidation of the o-substituted compound, whereas that of the p-halobenzaldehyde increases due to the increased inductive effect. The positional specificity was not due to electronic effects, because the oxidation rate was also decreased with electron-donating o-substituents. Although the substrates of aldehyde oxidase have not been so rigourously examined, the enzyme does appear to be subject to similar steric considerations, as o-chloro- and o-nitrobenzaldehyde are oxidized at much lower rates than benzaldehyde itself [33]. 

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. 

Substrate specificities can be broad and overlapping both among CYP family members and between CYPs and FMOs, and since metabolic transformations are often sequential (e.g. aliphatic hydroxylation being followed by oxidation by alcohol dehydrogenase, further oxidation to the acid, etc.), many enzymes and many metabolites can be involved in processing a single drug. Only a few of the most important enzymes involved in Phase I transformations have been mentioned here. For these and many others (monoamine oxidase, xanthine oxidase, etc.), further information can be found in the previously cited reviews. Bear in mind too that not all Phase I reactions are oxidative enzymes like carbonyl reductases are important in metabolism as well. 

Xanthina oxidase, xanthine dehydrogenase, Schardtnger enzyme an enzyme of aerobic purine degradation, which catalyses the oxidation of hypoxan-thine to xanthine, and xanthine to uric acid Hypox-anthine + HjO + 62 -> Xanthine -h H2O2 Xanthine + H2O -H O2 -> Uric acid + H2O2. It is a dimeric enzyme, M, 275,000, pH-optimum 4.7, pi 5.35, containing 2 FAD, 2 Mo and 8 Fe (data for the enzyme from milk). The substrate specificity is low it catalyses the oxidation of other purines (e. g. adenine), aU-phatic and aromatic aldehydes, pyrimidines, pteri-dines and other heterocyclic compounds. 

The specificity of this enzyme is low it will convert adenine to its 8-hydroxy and 2,8-dihydroxy derivatives and will also oxidize a wide variety of nonpurine substrates. Xanthine oxidase contains 2 moles of FAD, 2 moles of Mo(V), and 8 moles of Fe + its molecular weight is 200,000-... 

Greenlee L., Handler Xanthine Oxidase. Influence of pH on Substrate Specificity. The Journal of Biological Chemistry 239 1090-1095, 1964. 

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