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 C]urates 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.