Steady state kinetics of 4-hydroxyphenylpyruvate dioxygenase from human liver (III)

Kinetic experiments have been made with an apparently homogenous preparation of human liver 4-hydroxyphenylpyruvate dioxygenase Form 3 (4-hydroxyphenylpyruvate: oxygen oxidoreductase (hydroxylating, decarboxylating), EC 1.13.11.27) at 37 degrees in 0.2 M Tris/HCL, pH 7.5, by measuring the evolved carbon dioxide from the 1-14C-labeled substrate or the formation of homogentisate from the U-14C-labeled substrate. The effect of variations in the concentrations of substrates, products, and metal chelators on the velocity of the forward reaction was studied. The results agree with an Ordered Bi Bi kinetic mechanism (Cleland, W. W. (1963) Biochim. Biophys. Acta 67, 104--137), where 4-hydroxyphenylpyruvate is added prior to oxygen and CO2 released before homogentisate. A Theorell-Chance mechanism has not been excluded.