Purification, characterization, and kinetics of porcine recombinant dihydropyrimidine dehydrogenase.
Porcine recombinant dihydropyrimidine dehydrogenase was purified from Escherichia coli cells using cell disruption, ammonium sulfate fractionation, and chromatography on DEAE-cellulose and 2',5'-ADP-Sepharose. The yield was 60% with a specific activity of 14 units/mg protein. On SDS/PAGE the purified dehydrogenase exhibits a single band, indicating that no proteolytic degradation was taking place during purification. In agreement with the native enzyme, all cofactors, FMN, FAD, NADPH, and two iron-sulfur clusters, have been found. EPR spectra of the reduced dehydrogenase obtained at pH 9.5 are characteristic for two [4Fe-4S]1+ cubanes in dipolar interaction. Quantification of the observed signals indicated 0.95 spins per subunit, showing only partially reduced iron-sulfur clusters. The kinetic parameters of the porcine recombinant enzyme are very similar to those of the native enzyme. Thus, it can be concluded that the porcine recombinant enzyme behaves like the native dehydrogenase.
Rosenbaum
K
K
Theodor-Boveri-Institut für Biowissenschaften, Universität Würzburg, Germany.
Schaffrath
B
B
Hagen
W R
WR
Jahnke
K
K
Gonzalez
F J
FJ
Cook
P F
PF
Schnackerz
K D
KD
D000818:Animals; D001667:Binding, Competitive; D042943:Dihydrouracil Dehydrogenase (NADP); D004578:Electron Spin Resonance Spectroscopy; D005415:Flavins / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000032:analysis; D007700:Kinetics; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification*; D013440:Sulfides / Q000032:analysis; D013552:Swine; D014498:Uracil / Q000031:analogs & derivatives / Q000378:metabolism
D005415:Flavins; D011994:Recombinant Proteins; D013440:Sulfides; C007419:dihydrouracil; D014498:Uracil; D007501:Iron; D010088:Oxidoreductases; D042943:Dihydrouracil Dehydrogenase (NADP)