Molybdenum hydroxylases, oxidation

Electron density calculations suggest that electrophilic attack in pyridine (42) is favored at C-3, whereas nucleophilic attack occurs preferentially at C-2 and to a lesser extent at C-4. Cytochrome P-450 mediated ring hydroxylation of pyridine would, therefore, be expected to occur predominantly at C-3, the most electron-rich carbon atom. Although 3-hydroxypyridine is an in vivo metabolite in several species, the major C-oxidation product detected in the urine of most species examined was 4-pyridone (82MI10903). The enzyme system catalyzing the formation of this latter metabolite may involve the molybdenum hydroxylases and not cytochrome P-450 (see next paragraph). In the related heterocycle quinoline (43), positions of high electron density are at C-3, C-6 and C-8, while in isoquinoline (44) they are at C-5, C-7 and C-8. Nucleophilic substitution predictably occurs... 

In addition to these classical aromatic ring hydroxylations, many nitrogen heterocycles are substrates for molybdenum-containing enzymes, such as xanthine oxidase and aldehyde oxidase, which are present in the hepatic cytosolic fractions from various animal species. The molybdenum hydroxylases (B-75MI10902) catalyze the oxidation of electron-deficient carbons in aromatic nitrogen heterocycles. The reactions catalyzed by these enzymes are generally represented by equations (2) and (3). 

Molybdenum Hydroxylases (Aldehyde Oxidase, Xanthine Oxidase) Oxidations Purines, pteridine, methrotrexate, quinolones, 6-deoxycyclovir... 

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... 

Other enzymes may also be involved in the oxidation of aldehydes, particularly aldehyde oxidase and xanthine oxidase, which belong to the molybdenum hydroxylases. These enzymes are primarily cytosolic, although microsomal aldehyde oxidase activity has been detected. They are flavoproteins, containing FAD and also molybdenum, and the oxygen incorporated is derived from water rather than molecular oxygen. Aldehyde oxidase and xanthine oxidase in fact oxidize a wide variety of substrates, both aldehydes and nitrogen containing heterocycles such as caffeine and purines (see below). Aldehyde... 

Molybdenum hydroxylases (i.e., AO and XO) are flavoproteins that contain in addition to a FAD, a pterine cofactor coordinated to a molybdenum atom, and an iron sulfur center for their catalytic activity. They catalyze the two-electron oxidation of substrates with transfer to molecular oxygen to produce H2O2, and insert an atom of oxygen from water into a wide range of N-hctcrocycies and aldehydes via two-electron redox reaction as shown in equation 1.4 ... 

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. 

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]. 

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. 

In spite of the close structural relationship of the molybdenum hydroxylases, including a tendency for hydrophobic substrate/enzyme interaction, there is a very significant difference in the substrate specificity of the two enzymes. Not only is there considerable variation in the affinities for substrates and inhibitors, but there is often a difference in the position of oxidative attack. As both enzymes catalyse apparently similar nucleophilic reactions, this difference cannot be explained solely from electronic considerations and is probably due, to a great extent, to the differential response of each enzyme to steric factors. 

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)... 

In contrast, pteridines are excellent substrates for both molybdenum hydroxylases and each enzyme can catalyse oxidation at carbon 2, 4 or 7, although not necessarily in the same molecule [ 10, 204-206 ]. For a comparison of the position of attack by the enzymes, the reader is referred to a recent review by the author [11]. Unfortunately, the sequence of oxidation in the multistep pathways is not always clear and therefore the effects of substitution are difficult to interpret. l-Methylpteridin-4-one (55b), which is apparently oxidized at carbon 2, is converted much more rapidly than the 3-methyl isomer (55a) [206], These derivatives correspond to the 3-methyl and 1-methyl analogues of hypoxanthine, respectively. Nevertheless, it is not possible to make direct comparisons of the //-methyl derivatives with unsubstituted pteridin-4-one (56b) because the site of oxidation may differ [206, 207],... 

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