Structural analysis of urate oxidase in complex with its natural substrate inhibited by cyanide: Mechanistic implications

The catalytic mechanism was investigated through a ternary complex formed between the enzyme, uric acid, and cyanide that stabilizes an intermediate state of the reaction. When uric acid is replaced by a competitive inhibitor, no complex with cyanide is formed.The X-ray structure of this compulsory ternary complex led to a number of mechanistic evidences that support a sequential mechanism in which the two reagents, dioxygen and a water molecule, process through a common site located 3.3 ？ above the mean plane of the ligand. This site is built by the side chains of Asn 254, and Thr 57, two conserved residues belonging to two different subunits of the homo-tetramer. The absence of a ternary complex between the enzyme, a competitive inhibitor, and cyanide suggests that cyanide inhibits the hydroxylation step of the reaction, after the initial formation of a hydroperoxyde type intermediate.Urate oxidase (uricase; EC 1.7.3.3; or UOX) belongs to the purine degradation pathway and catalyzes in the presence of molecular oxygen the hydroxylation of uric acid into a metastable product identified as the 5-hydroxyisourate (5-HIU) [1]. Once released in solution, 5-HIU decays slowly to allantoin, a process independent of oxygen and associated with the release of CO2 (dehydro-decarboxylation).). In vivo, 5-HIU is rapidly processed by two specific enzymes to [S]-allantoin [2,3], the structures of which were solved [4,5]. Urate oxidase is present in many species, but is absent in human and higher apes. This emphasizes an evolutionary advantage since it was suggested that uric acid being a powerful anti-oxidant, humans would have less free radicals, so less cancer due to aging. As a consequence, uric acid level in plasma is quite elevated and a higher pathological level may be fatal. Sanofi-Aventis produces and commercializes urate oxidase, first extracted from Aspergillus flavus and now expressed in Saccharomyces cerevisiae, branded under the name Fasturtec？ (DCI : Rasburicase), to