Enzymes xanthine dehydrogenase

Example of a Protein Purification Scheme Purification of the Enzyme Xanthine Dehydrogenase from a Eungus... 

Most purification procedures for a particular protein are developed in an empirical manner, the overriding principle being purification of the protein to a homogeneous state with acceptable yield. Table 5.5 presents a summary of a purification scheme for a selected protein. Note that the specific activity of the protein (the enzyme xanthine dehydrogenase) in the immuno-affinity purified fraction (fraction 5) has been increased 152/0.108, or 1407 times the specific activity in the crude extract (fraction 1). Thus, xanthine dehydrogenase in fraction 5 versus fraction 1 is enriched more than 1400-fold by the purification procedure. 

As noted earlier, peroxynitrite is formed with a diffusion-controlled rate from superoxide and nitric oxide (Reaction 10). As both these radicals are ubiquitous species, which present practically in all cells and tissues, peroxynitrite can be the most important species responsible for free radical-mediated damage in biological systems. Moreover, it is now known that NO synthases are capable of producing superoxide and nitric oxide simultaneously (see Chapter 22), greatly increasing the possible rate of peroxynitrite production. In addition, another enzyme xanthine dehydrogenase is also able to produce peroxynitrite in the presence of nitrite... 

The cytosolic enzyme xanthine dehydrogenase catalyses the oxidation of hypoxanthine and xanthine to uric acid. It is thought to be located predominantly in the liver, small intestine and capillary endothelium in man [9]. However, the distribution is different in other species. In healthy tissue, most of the enzyme is present as the D form , which transfers electrons to NAD+ ... 

Under conditions of stress, the enzyme xanthine dehydrogenase is converted to xanthine oxidase, which uses 2 O2 instead of NAD to oxidize hypoxanthine and xanthine, and therefore releases 2O2. Thus, inhibitors of xanthine oxidase such as allopurinol are used to suppress inflammation associated with arthritis. (Xanthine oxidase is discussed in more detail in Chapter 8.18)... 

Figure 1.14. Examples of the effects of a mutation. The mutation rosy in Drosophila is lacking the enzyme xanthine dehydrogenase. Therefore, at least three compounds are not synthesized. Arrows show the actions catalyzed by the enzyme.

Molybdenum was long known as essential for the growth of all higher plants. Then, in 1953 it was found in the essential enzyme, xanthine dehydrogenase. 

Xanthine dehydrogenase that mediates the conversion of hypoxanthine into xanthine and uric acid has been studied extensively since it is readily available from cow s milk. It has also been studied (Leimkiihler et al. 2004) in the anaerobic phototroph Rhodobacter capsulatus, and the crystal structures of both enzymes have been solved. Xanthine dehydrogenase is a complex flavoprotein containing Mo, FAD, and [2Fe-2S] redox centers, and the reactions may be rationalized (Hille and Sprecher 1987) ... 

Although it had been assumed that only hypoxanthine dehydrogenase is required for the conversion of hypoxanthine (6-hydroxypurine) into uric acid, in Clostridium purinolyti-cum, two enzymes, both of which contain a selenium cofactor, are required. The enzymes differ in the molecular mass of their subunits, in their terminal amino acid sequences, in their kinetic parameters, and in their specific activities for purines (Self and Stadman 2000). Purine hydroxylase converts purine into hypoxanthine and xanthine (2,6-dihy-droxypurine), which is then further hydroxylated to uric acid (2,6,8-trihydroxypurine) by xanthine dehydrogenase (Self 2002). 

Koenig K, JR Andreesen (1990b) Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas putida Fnl two molybdenum-containing enzymes of novel structural composition. JBacteriol 111 5999-6009. 

Furthermore, depletion of hepatic GSH induced chemically or by fasting augmented hepatic I/R-induced enzyme release and promoted lipid peroxidation (Jennische, 1984 Stein et al., 1991) Benoit et al. (1992) have used portacaval-shunted rats as a model of chronic hepatic ischaemia, and were able to show decreases in total levels of SOD and xanthine dehydrogenase, but no significant change in catalase or glutathione peroxidase. 

Of the mammalian enzymes, the sulphite oxidase of bovine liver has only recently been discovered to contain molybdenum (15). The better known molybdenum enzymes, xanthine oxidase from cows milk (31) and aldehyde oxidase from rabbit liver (16) are closely related to one another as they are to the xanthine dehydrogenases from chicken liver (17) and from bacteria (18). 

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. 

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). 

This enzyme [EC 1.1.3.22] catalyzes the reaction of xanthine with dioxygen and water to produce urate and hydrogen peroxide. Enzymatic activity requires iron, FAD, and molybdenum. Hypoxanthine and some other purines and pterins can act as substrates. Under some conditions, the product is mainly superoxide rather than hydrogen peroxide thus, R—H reacts with two dioxygen and water to produce R—OH, two H+, and two 02 molecules. The Micrococcus enzyme can use ferredoxin as the acceptor substrate. The mammalian enzyme can be interconverted to xanthine dehydrogenase [EC 1.1.1.204]. See Xanthine Dehydrogenase... 

Pyridine nucleotide-dependent flavoenzyme catalyzed reactions are known for the external monooxygenase and the disulfide oxidoreductases However, no evidence for the direct participation of the flavin semiquinone as an intermediate in catalysis has been found in these systems. In contrast, flavin semiquinones are necessary intermediates in those pyridine nucleotide-dependent enzymes in which electron transfer from the flavin involves an obligate 1-electron acceptor such as a heme or an iron-sulfur center. Examples of such enzymes include NADPH-cytochrome P4S0 reductase, NADH-cytochrome bs reductase, ferredoxin — NADP reductase, adrenodoxin reductase as well as more complex enzymes such as the mitochondrial NADH dehydrogenase and xanthine dehydrogenase. 

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