IDENTIFICATION OF THE TRUE PRODUCT OF THE URATE OXIDASE REACTION

The O-2-dependent oxidation of urate catalyzed by urate oxidase has be en examined in order to identify the immediate product of the enzymati c reaction. Specifically labeled [C-13]urates were utilized as substra tes, and the time courses were monitored by C-13 NMR. On the basis of chemical shift values and O-18 labeling, the product of the reaction w as identified as 5-hydroxyisourate. This identification was substantia ted by calculation of the C-13 NMR spectrum of 5-hydroxyisourate using ab initio density functional theory methods. The predominant tautomer s of urate and allantoin in aqueous solution were identified from C-13 NMR titration data; the ionization behavior of urate and 5-hydroxyiso urate were also examined by computational methods. The nonenzymatic pa thway for production of allantoin from 5-hydroxyisourate was delineate d; the reaction proceeds by the hydrolysis of the N1-C6 bond, followed by an unusual 1,2-carboxylate shift and decarboxylation to form allan toin.