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


CONCLUSIONS
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 A 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.
Gabison
Laure
L
LCRB, UMR CNRS 8015, Université Paris Descartes, 4 avenue de l'Observatoire, 75270 Paris cedex 06, France. laure.gabison@univ-paris5.fr
Prangé
Thierry
T
Colloc'h
Nathalie
N
El Hajji
Mohamed
M
Castro
Bertrand
B
Chiadmi
Mohamed
M
D001231:Aspergillus flavus / Q000201:enzymology*; D002384:Catalysis / Q000187:drug effects; D018360:Crystallography, X-Ray; D003486:Cyanides / Q000737:chemistry / Q000494:pharmacology*; D055672:Static Electricity; D013379:Substrate Specificity / Q000187:drug effects; D014503:Urate Oxidase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D014527:Uric Acid / Q000737:chemistry
D003486:Cyanides; D014527:Uric Acid; D014503:Urate Oxidase