Xanthine oxidase active site structure

The ability of quinazoline to enter the xanthine oxidase active site, and the presence of an active site nucleophile near the alkylating center, very likely accounts for enzyme inactivation by these hydroquinones. The quinone (696) acts as an oxidizing suicide substrate. It is able to enter the xanthine oxidase active-site, and by the enzyme reductive action and HCl elimination it becomes a reductive alkylating agent (698) targeted towards cellular structures important to the cancer cell <88JOC6099>. 

Figure 17.2 The structure of the pterin cofactor (1) which is common to most molybdenum- and tungsten-containing enzymes and schematic active site structures for members of the xanthine oxidase (2,3), sulfite oxidase (4) and DMSO reductase (5-7) enzyme families. (From Enemark et al., 2004. Copyright (2004) American Chemical Society.)...

Figure 16. Consensus oxidized active-site structures of the xanthine oxidase (XO), sulfite oxidase (SO), and DMSO reductase (DMSOR), and aldehyde oxidoreductase (AOR) families of mononuclear molybdenum and tungsten enzymes and the structure of the common ppd cofactor (41, 42). The question mark in the AOR structure indicates uncertainty in the presence of a coordinated water molecule.

Molybdenum-containing enzymes can be divided into three families, the xanthine oxidase (XO), sulfite oxidase (SO), and the DMSO reductase (DMR) families. They each have a characteristic active site structure (Figure 17.2(a)) and catalyse a particular type of reaction (see below). Whereas in eukaryotes, the pterin side chain has a terminal phosphate group, in prokaryotes, the cofactor (R in Figure 17.2(b)) it is often a dinucleotide. 

Figure 16 The biosynthesis of Moco and bis-MGD. Shown is a scheme of the biosynthetic pathway for Moco biosynthesis in bacteria and eukaryotes. The proteins involved in the reactions are colored in red for bacterial proteins and blue for human proteins. In bacteria, Moco (54) can be further modified by the attachment of, for example, GMP, forming MGD, and two equivalents of MGDare bound to molybdenum, forming the so-called bis-MGD cofactor (56). Further, Moco can be modified by the replacement of one 0x0 ligand by a sulfido ligand, forming the monooxo Moco (55). The three molybdenum containing enzyme families are divided into the xanthine oxidase, sulfite oxidase, and DMSO reductase families according to their active site structures.

Figure 6.1 Schematic active site structures of DMSO reductase (left), sulfite oxidase (middle) and xanthine oxidase (right) families, and the structure of pyranopterindithiolate (MPT).

XOR accelerates the hydroxylation of purines, pyrimidines, pterins and aldehydes [132]. In humans, the enzyme catalyzes the last two steps of purine catabolism the oxidation of hypoxanthine to xanthine and of the latter to uric acid. An unusual property of this, but not aU XOR enzymes [133], is its interconversion between xanthine dehydrogenase and xanthine oxidase activities which implies a switch between NAD" and molecular oxygen being used as the final electron acceptor [134]. Structural studies suggest that this switch, that can be irreversibly induced by proteolysis [135], results from conformational changes that lead to both restricted access to the NAD cofactor to its binding site and changes in the redox potential of the FAD cofactor [136],... 

RGURE 22-47 Allopurinol, an inhibitor of xanthine oxidase. Hypoxanthine is the normal substrate of xanthine oxidase. Only a slight alteration in the structure of hypoxanthine (shaded pink) yields the medically effective enzyme inhibitor allopurinol. At the active site, allopurinol is converted to oxypurinol, a strong competitive inhibitor that remains tightly bound to the reduced form of the enzyme. 

Calculations on xanthine oxidase-like active sites have been carried out by Bray and Deeth and by Voityuk et al. using density functional (DF) approaches [216-218,220], Bray and Deeth show that the LMoOS(OH) structure is quite favorable for the Mo(VI) state of xanthine oxidase. Five-coordination in the... 

The SOD activity measurements using the xanthine-xanthine oxidase nitro blue tetrazolium (NBT) method [45] indicated that a concentration of 0.75 xM of [Mn(OBz)(H-3,5-/-Pr2pz)(L 1)] 1 and 0.8 iM of the related [Mn(OBz)(Ll)] 2 complex exert the same SOD activity as one unit of native SOD. The high SOD activity of the tris(pyrazolyl)borate complexes was attributed to their structural similarities to the MnSOD active site [45],... 

A wide range of soluble redox enzymes contain one or more intrinsic [2Fe-2S]2+ +, [3Fe-4S]+ , or [4Fe S]2+ + clusters that function in electron transport chains to transfer electrons to or from nonheme Fe, Moco/Wco, corrinoid, flavin, thiamine pyrophosphate (TPP), Fe S cluster containing, or NiFe active sites. Many have been structurally and spectroscopically characterized and only a few of the most recent examples of each type are summarized here. Dioxygenases that function in the dihydroxylation of aromatics such as benzene, toluene, benzoate, naphthalene, and phthalate contain a Rieske-type [2Fe-2S] + + cluster that serves as the immediate electron donor to the monomeric nonheme Fe active site see Iron Proteins with Mononuclear Active Sites). The xanthine oxidase family of molybdoenzymes see Molybdenum MPT-containing Enzymes) contain two [2Fe-2S] + + clusters that mediate electron transfer between the Moco active site and the Other soluble molybdoen-... 

A (39). In addition, the Mo(V) EPR spectrum of arsenite-inhibited xanthine oxidase clearly shows hyperfine structure from the As nucleus (40,41). In binding at the molybdenum center, arsenite blocks the active site and inhibits electron transfer activity in xanthine oxidase via the effect it has on the relative reduction potentials of the redox-active centers (41). 

The initial contribution to this volume provides a detailed overview of how spectroscopy and computations have been used in concert to probe the canonical members of each pyranopterin Mo enzyme family, as well as the pyranopterin dithiolene ligand itself. The discussion focuses on how a combination of enzyme geometric structure, spectroscopy and biochemical data have been used to arrive at an understanding of electronic structure contributions to reactivity in all of the major pyranopterin Mo enzyme families. A unique aspect of this discussion is that spectroscopic studies on relevant small molecule model compounds have been melded with analogous studies on the enzyme systems to arrive at a sophisticated description of active site electronic structure. As the field moves forward, it will become increasingly important to understand the structure, function and reaction mechanisms for the numerous non-canonical [ie. beyond sulfite oxidase, xanthine oxidase, DMSO reductase) pyranopterin Mo enzymes. 

Enzymes from the DMSO reductase family are distinct in several respects from those found in the xanthine oxidase and sulfite oxidase families. Members of the DMSO reductase family are only found in prokaryotes. They are also perhaps the most difficult members of the Mo and W enzyme superfamily to classify as there is so much structural diversity both at the active site and... 

Xanthine oxidase (XO) catalyzes the hydroxylation of hypoxanthine to xanthine and xanthine to uric acid. It plays an important role in the catabolism of purines.As shown in Figure 7.11, this reaction is slightly more complicated than the pure oxo-transfer reaction catalyzed by the other two enzymes discussed in this review. The structure of the XO active site has also been more controversial than for the other two enzymes. However, using a combination of crystallographic, EXAFS and computational studies it has been shown that the oxidized state has the structure MoOS(OH)(MPT), with the 0x0 group in the axial position. " Extensive experimental and theoretical investigations have been performed on XO and related enzymes " and the latter are summarized in Table 7.3. 

---