Dehalogenating and NADPH-modifying activities of dihydropyrimidine dehydrogenase.
Dihydropyrimidine dehydrogenase (DPDase) catalyzed the debromination of 5-bromo-5,6-dihydrouracil (BrUH2) to uracil at pH 7.7 and 37 degrees C. The debrominating activity of DPDase was increased 5-fold by treatment with H2O2, whereas the dehydrogenating activity was inhibited by this treatment. The time course for increasing the debrominating activity by H2O2 was similar to that for decreasing the dehydrogenating activity. Thus, the relative amounts of debrominating and dehydrogenating activities of DPDase were reciprocally related. H2O2 treatment of DPDase decreased the number of thiol groups reactive with 5,5'-dithiobis(2-nitrobenzoate) from eight/subunit to less than one. The kcat for debromination of BrUH2 by H2O2-treated DPDase (OxDPDase) was 1.9 s-1, which was comparable with kcat for reduction of thymine (2.1 s-1) by DPDase. Even though the debromination of BrUH2 to uracil does not involve a net reduction of BrUH2, NADPH was required for this activity. The reaction of OxDPDase with 5-iodo-5,6-dihydrouracil (IUH2) was more complicated than that with BrUH2. Aerobically, OxDPDase catalyzed the deiodination of IUH2 to uracil and the iodination of NADPH to 5-iodo-6-hydroxy-1,4,6-trihydronicotinamide adenine dinucleotide phosphate. The turnover number for the iodination reaction was enhanced by NaI and had a value of 3.5 s-1 in the presence of 4 mM IUH2 and 50 mM NaI. Anaerobically, OxDPDase catalyzed the above reactions, the deiodination of IUH2 to 5,6-dihydrouracil, and the hydration of NADPH to 6-hydroxy-1,2,3,4-tetrahydronicotinamide adenine dinucleotide phosphate. The turnover number for the anaerobic hydration of NADPH was similar to that for the aerobic iodination of NADPH.
Porter
D J
DJ
Division of Experimental Therapy, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
D000818:Animals; D001966:Bromine / Q000378:metabolism*; D002417:Cattle; D042943:Dihydrouracil Dehydrogenase (NADP); D006861:Hydrogen Peroxide / Q000378:metabolism; D006865:Hydrogenation; D007455:Iodine / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014498:Uracil / Q000031:analogs & derivatives / Q000378:metabolism
C049571:5-bromo-5,6-dihydrouracil; C073482:eniluracil; D009249:NADP; D014498:Uracil; C013573:5-iodo-5,6-dihydrouracil; D007455:Iodine; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D042943:Dihydrouracil Dehydrogenase (NADP); D001966:Bromine