SECONDARY TRITIUM AND SOLVENT DEUTERIUM-ISOTOPE EFFECTS AS A PROBE OFTHE REACTION CATALYZED BY PORCINE RECOMBINANT DIHYDROPYRIMIDINE DEHYDROGENASE

Dihydropyrimidine dehydrogenase catalyzes the rate-limiting step in th e degradation of pyrimidines in mammals, the reduction of uracil or th ymine to their 5,6-dihydro derivatives. The reduction of uracil by enz yme-bound reduced flavin involves both proton and hydride transfer. In order to determine whether hydride and proton transfer occur in a con certed or stepwise fashion, and to determine the nature of the transit ion state for the reduction, secondary tritium kinetic isotope effects were measured in H2O and D2O. The tritium isotope effect using 5-H-3- uracil is 0.90 +/- 0.03 in H2O and becomes more inverse, 0.85 +/- 0.04 , in D2O. Data are interpreted in terms of a stepwise reduction at C-6 followed by protonation at C-5. A late transition state is proposed f or the proton transfer at C-5 of uracil.