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7 em a /em ). site of the enzyme is absent in the present structure, which probably accounts for the stability of this intermediate toward ligand displacement by hydroxide. Molybdenum hydroxylases are found in virtually all organisms, ranging from bacteria to humans, and catalyze the hydroxylation of a wide variety of heterocyclic substrates such as purines, pyrimidines, and pterins, in addition to aldehydes (1, 2). The overall reaction generates rather than consumes reducing equivalents, which sets it apart from other hydroxylation systems, such as the cytochromes P450. Xanthine oxidoreductase (XOR) is the prototypical molybdenum hydroxylase and, in humans, it catalyzes the hydroxylation of hypoxanthine to xanthine as well as xanthine to uric acid. The enzyme is a well established target of drugs against gout and hyperuricemia (3). XOR from bovine milk, the most studied molybdenum hydroxylase, has served as a benchmark for the entire class of complex metalloflavoproteins (4). Each subunit of the homodimeric enzyme contains one molybdenum center, one flavin (FAD), and two [2FeC2S] iron-sulfur centers (1, 2). The oxidative hydroxylation of xanthine to uric acid takes place at the molybdenum center and results in the two-electron reduction of the metal from Mo(VI) to Mo(IV). The enzyme is subsequently reoxidized by NAD+ or molecular oxygen (1) in a reaction that occurs at the FAD (after electron transfer from the molybdenum center via the iron-sulfur centers). The mammalian enzyme exists in two alternative forms of the same gene product. Under normal circumstances, it occurs in its dehydrogenase form [xanthine dehydrogenase (XDH)], but it can be readily converted to its oxidase form (XO) by oxidation of sulfhydryl residues or by limited proteolysis (1). XDH shows a preference for NAD+ as the oxidizing substrate (although it is also able to Lercanidipine react with O2), whereas XO is unable to react with NAD+ and uses dioxygen exclusively (1). X-ray crystal structures of both the XDH and XO forms of bovine XOR have recently been reported at 2.1- and 2.5-? resolution (5), respectively, and a detailed comparison of Lercanidipine the two forms and site-directed mutagenesis studies has elucidated the structural basis for the XDH to XO switch (6). Based on the results of extended x-ray absorption fine-structure studies (2), two thiolene sulfurs (S), one sulfo (?S), one oxo (?O), and one hydroxo (OH) group or water had previously been postulated as the ligands of the active-site molybdenum center (Fig. 1(13), with minor modifications. Bound oxipurinol was released from the complex spontaneously by incubating the enzyme solution under air-saturated conditions without additional oxidizing reagent for 48 h at 25C. More than 95% of the purified enzyme was catalytically active (the activity to flavin ratio 200; ref. 13). Purified samples were stored on ice in a mixture of 80% 0.1 M pyrophosphate buffer (pH 8.5) and 20% 50 mM TrisHCl buffer (pH 7.8), both containing 0.2 mM EDTA and 1 mM salicylate. The XDH concentration was determined by using an extinction coefficient of 37,800 MC1cmC1 (14). 4-[5-Pyridin-4-yl-1H-[1,2,4]triazol-3-yl]pyridine-2-carbonitrile (FYX-051) and 2-hydroxy-FYX-051, shown in Fig. 1, were provided by Fuji Yakuhin, Tokyo. Titanium(III) citrate solutions were prepared in an argon glove box (1ADB-2, MIWA, Osaka) (15, 16). All other reagents were of the highest grade commercially available. The compound released from the complex generated on mixing XDH Rabbit Polyclonal to c-Jun (phospho-Tyr170) with FYX-051 was identified as follows. The sample was mixed with a 6-fold excess volume of methanol and stored on ice before HPLC analysis on an RP-18-GP column Lercanidipine (Kanto, Tokyo). It was eluted with 80% of 0.5% acetic acid and 20% acetonitrile at 40C. Elution profiles were monitored at 277 nm. Liquid chromatography/MS was performed with an API150EX apparatus (Applied Biosystems). Enzyme Assay and Analytical Procedures for the XDHCFYX-051 Complex. Xanthine-O2 activity was determined in a solution containing 0.15 mM xanthine, 0.1 M pyrophosphate buffer (pH 8.5), and 0.2 mM EDTA under air-saturated conditions, following the rate of uric acid formation at 295 nm at 25C by using a Hitachi (Tokyo) U3300.