Tags

Type your tag names separated by a space and hit enter

Purification and characterization of dihydropyrimidine dehydrogenase from Alcaligenes eutrophus.
Arch Biochem Biophys. 1996 Aug 01; 332(1):175-82.AB

Abstract

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].

Authors+Show Affiliations

Theodor-Boveri-Institut für Biowissenschaften, Physiologische Chemie I, Würzburg, Germany.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

8806723

Citation

Schmitt, U, et al. "Purification and Characterization of Dihydropyrimidine Dehydrogenase From Alcaligenes Eutrophus." Archives of Biochemistry and Biophysics, vol. 332, no. 1, 1996, pp. 175-82.
Schmitt U, Jahnke K, Rosenbaum K, et al. Purification and characterization of dihydropyrimidine dehydrogenase from Alcaligenes eutrophus. Arch Biochem Biophys. 1996;332(1):175-82.
Schmitt, U., Jahnke, K., Rosenbaum, K., Cook, P. F., & Schnackerz, K. D. (1996). Purification and characterization of dihydropyrimidine dehydrogenase from Alcaligenes eutrophus. Archives of Biochemistry and Biophysics, 332(1), 175-82.
Schmitt U, et al. Purification and Characterization of Dihydropyrimidine Dehydrogenase From Alcaligenes Eutrophus. Arch Biochem Biophys. 1996 Aug 1;332(1):175-82. PubMed PMID: 8806723.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Purification and characterization of dihydropyrimidine dehydrogenase from Alcaligenes eutrophus. AU - Schmitt,U, AU - Jahnke,K, AU - Rosenbaum,K, AU - Cook,P F, AU - Schnackerz,K D, PY - 1996/8/1/pubmed PY - 1996/8/1/medline PY - 1996/8/1/entrez SP - 175 EP - 82 JF - Archives of biochemistry and biophysics JO - Arch Biochem Biophys VL - 332 IS - 1 N2 - 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]. SN - 0003-9861 UR - https://www.unboundmedicine.com/medline/citation/8806723/Purification_and_characterization_of_dihydropyrimidine_dehydrogenase_from_Alcaligenes_eutrophus_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-9861(96)90330-6 DB - PRIME DP - Unbound Medicine ER -