Purification and properties of dihydrothymine dehydrogenase from rat liver.
Rat liver dihydrothymine dehydrogenase, the rate-limiting enzyme of thymidine and uridine degradation, was purified to homogeneity as judged by polyacrylamide disc gel electrophoresis, sedimentation velocity, and Ultrogel ACA-34 elution profile. The enzyme has a molecular weight of 220,000 +/- 5,000 as determined by Ultrogel ACA-34 and sedimentation equilibrium The s20,w value of the enzyme was 9.2 S. The isoelectric point was at pH 5.25. The enzyme is composed of two identical subunits of an approximate molecular weight of 110,000 +/- 3,000 as determined by sodium dodecyl sulfate disc gel electrophoresis. The enzyme contains 4 mol of FAD and 3 mol of iron per mol of enzyme. Flavin released from the enzyme by boiling was identified as FAD by absorption spectra and thin layer chromatography, indicating that the enzyme is a flavometal protein. During dialysis, the enzyme was stabilized by 2-mercaptoethanol, but neither NADPH nor thymine was effective. The relative rates of reduction of pyrimidine analogues substituted at position 5 were 5-fluorouracil > 5-bromouracil > 5-diazouracil > 5-iodouracil > 5-nitrouracil, with 5-fluorouracil and 5-diazouracil 70% faster than thymine. Uracil was reduced 25% faster than thymine. The pH optimum for the forward and reverse reactions was 7.4. In the presence of NADPH, the apparent Km was 2.6 microM for thymine and 1.8 microM for uracil. Apparent Km for NADPH was 15 microM with thymine as substrate and 11 microM with uracil. In the reverse reaction, apparent Km values were 43 microM for dihydrothymine and 193 microM for dihydrouracil; apparent Km for NADP+ was 3.8 microM with dihydrothymine as substrate and 2.9 microM with dihydrouracil.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D042943:Dihydrouracil Dehydrogenase (NADP); D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D013053:Spectrophotometry; D013379:Substrate Specificity; D014498:Uracil / Q000302:isolation & purification / Q000378:metabolism
D000596:Amino Acids; D005182:Flavin-Adenine Dinucleotide; D014498:Uracil; D010088:Oxidoreductases; D042943:Dihydrouracil Dehydrogenase (NADP)