Identification of the True Product of the Urate Oxidase Reaction

The O 2 -dependent oxidation of urate catalyzed by urate oxidase has been examined in order to identify the immediate product of the enzymatic reaction. Specifically labeled 13 Curates were utilized as substrates, and the time courses were monitored by 13 C NMR. On the basis of chemical shift values and 18 O labeling, the product of the reaction was identified as 5-hydroxyisourate. This identification was substantiated by calculation of the 13 C NMR spectrum of 5-hydroxyisourate using ab initio density functional theory methods. The predominant tautomers of urate and allantoin in aqueous solution were identified from 13 C NMR titration data; the ionization behavior of urate and 5-hydroxyisourate were also examined by computational methods. The nonenzymatic pathway for production of allantoin from 5-hydroxyisourate was delineated; the reaction proceeds by the hydrolysis of the N1−C6 bond, followed by an unusual 1,2-carboxylate shift and decarboxylation to form allantoin.