Tritium isotope effects in the reaction catalyzed by 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. strain P.J. 874

Abstract

Tritium isotope effects in the reaction catalyzed by 4-hydroxyphenylpyruvate dioxygenase (4-hydroxyphenylpyruvate:oxygen oxidoreductase (hydroxylating, decarboxylating), EC 1.13.11.27) from Pseudomonas sp.l strain P.J. 874 were studied with 14C and different 3H-labelled 4-hydroxyphenylpyruvate. Tritium of ring-2,6- 3H 2-1abelled substrate was released into water in 1:2 stoichiometry to 14CO 2 formation. The tritium release from ring-3,5- 3H 2- and side chain-3- 3H 1-labelled 4-hydroxyphenylpyruvate was low as compared with 14CO 2 formation. The apparent tritium isotope effects were below two, as judged by comparison of 3H/ 14C ratios of 4-hydroxyphenylpruvate and homogentisate. The ratios showed no dependence on oxygen concentrations between 1 and 21% in the gas phase. Thus, a tritium assay can be used to determine the activity of 4-hydroxyphenylpyruvate dioxygenase. Apparently, none of the substrate hydrogens is involved in any rate-limiting step up to the first irreversible step. enol-4-Hydroxyphenylpyruvate was excluded as the active substrate tautomer.