PURIFICATION AND CHARACTERIZATION OF DIHYDROPYRIMIDINE DEHYDROGENASE FROM ALCALIGENES-EUTROPHUS

Dihydropyrimidine dehydrogenase from Alcaligenes eutrophus was purifie d to homogeneity using ammonium sulfate fractionation and chromatograp hy on phenyl-Sepharose, MonoQ-Sepharose, and 2,5-ADP-Sepharose. The en zyme is a homotetramer with a subunit molecular mass of 52 kDa, The ab sorption spectrum of the bacterial dihydropyrimidine dehydrogenase has maxima in the 300- and 400-nm region, suggesting a flavoprotein. The enzyme contains 4 mol FMN, about 24 mol iron and acidlabile sulfide pe r mole of protein, implying a flavoprotein with FeS centers. The bacte rial dehydrogenase is NADPH dependent with B-side stereospecificity. T he initial velocity patterns of the bacterial dehydrogenase together w ith isotope exchange at equilibrium and a quantitative analysis of the product and dead-end inhibition data suggest a rapid equilibrium rand om kinetic mechanism, which is in contrast to results obtained for dih ydropyrimidine dehydrogenase from pig liver. The pig liver enzyme adhe res to a nonclassical two-site ping-pong kinetic mechanism [B. Podschu n, P. F. Cook, and K. D. Schnackerz (1990) J. Biol. Chem. 265, 12966-1 2972], whereas for the bovine enzyme a rapid equilibrium random kineti c mechanism was proposed based on steady-state kinetic data [D. J. T. Porter and T. Spector (1993) J. Biol. Chem. 268, 19321-19327]. (C) 199 6 Academic Press, Inc.