Urate oxidase: Single-turnover stopped-flow techniques for detecting two discrete enzyme-bound intermediates

Abstract

Urate oxidase is a true oxidase; although the product is hydroxylated, the oxygen in the hydroxyl group derives from solvent, and one equivalent of dioxygen is reduced to H 2 0 2 concomitant with urate oxidation. In leguminous plants, urate oxidase is a constituent of the ureide pathway by which fixed nitrogen is converted to allantoin and allantoate, the metabolites that serve to transport nitrogen from the roots to the stems, leaves, and seed pods of the plant. In mammals urate oxidase facilitates the excretion of excess nitrogen by virtue of the greater solubility of allantoin relative to urate. Urate oxidase is also found in a wide variety of microorganisms, and some bacteria can grow on urate as their sole carbon and nitrogen source. The goals of these studies were both qualitative and quantitative. In particular, the mechanism illustrated in the chapter proposes the formation of three discrete intermediates in the course of the catalytic reaction, and stopped-flow studies were conducted to determine whether evidence could be adduced for their existence. An alternative mechanism for the urate oxidase reaction has been proposed that suggests that oxidation of urate occurs without formation of any discrete intermediates.