Purification and characterization of dihydropyrimidine dehydrogenase from Alcaligenes eutrophus.
Dihydropyrimidine dehydrogenase from Alcaligenes eutrophus was purified to homogeneity using ammonium sulfate fractionation and chromatography on phenyl-Sepharose, MonoQ-Sepharose, and 2,5-ADP-Sepharose. The enzyme is a homotetramer with a subunit molecular mass of 52 kDa. The absorption 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 per mole of protein, implying a flavoprotein with FeS centers. The bacterial dehydrogenase is NADPH dependent with B-side stereospecificity. The initial velocity patterns of the bacterial dehydrogenase together with isotope exchange at equilibrium and a quantitative analysis of the product and dead-end inhibition data suggest a rapid equilibrium random kinetic mechanism, which is in contrast to results obtained for dihydropyrimidine dehydrogenase from pig liver. The pig liver enzyme adheres to a nonclassical two-site ping-pong kinetic mechanism [B. Podschun, P. F. Cook, and K. D. Schnackerz (1990) J. Biol. Chem. 265, 12966-12972], whereas for the bovine enzyme a rapid equilibrium random kinetic mechanism was proposed based on steady-state kinetic data [D. J. T. Porter and T. Spector (1993) J. Biol. Chem. 268, 19321-19327].
Schmitt
U
U
Theodor-Boveri-Institut für Biowissenschaften, Physiologische Chemie I, Würzburg, Germany.
Jahnke
K
K
Rosenbaum
K
K
Cook
P F
PF
Schnackerz
K D
KD
D000421:Alcaligenes / Q000201:enzymology*; D000645:Ammonium Sulfate; D000818:Animals; D002417:Cattle; D005591:Chemical Fractionation; D002847:Chromatography, Agarose; D042943:Dihydrouracil Dehydrogenase (NADP); D004791:Enzyme Inhibitors / Q000494:pharmacology; D005420:Flavoproteins / Q000737:chemistry / Q000302:isolation & purification; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009249:NADP / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013053:Spectrophotometry; D013237:Stereoisomerism; D013552:Swine
D004791:Enzyme Inhibitors; D005420:Flavoproteins; D009249:NADP; D010088:Oxidoreductases; D042943:Dihydrouracil Dehydrogenase (NADP); D000645:Ammonium Sulfate