Background: Urate oxidase (EC 1.7.3.3 or UOX) catalyzes the conversion of uric acid and
gaseous molecular oxygen to 5-hydroxyisourate and hydrogen peroxide, in the absence of cofactor
or particular metal cation. The functional enzyme is a homo-tetramer with four active sites located
at dimeric interfaces.
Results: 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.
Conclusion: 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 Å 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.