Dihydropyrimidine dehydrogenase. Kinetic mechanism for reduction of uracil by NADPH.
Steady-state and pre-steady-state kinetic data were used to determine the kinetic mechanism for bovine liver dihydropyrimidine dehydrogenase (DPDase). Steady-state kinetic data suggested a random rapid-equilibrium mechanism with Km values for NADPH and uracil of 0.12 microM and 0.8 microM, respectively, and a kcat of 1.6 s-1 in Tris buffer at pH 8.0 and 37 degrees C. The dissociation constant of DPDase for NADPH at 25 degrees C in the absence of uracil (0.09 microM) was similar to the Km for NADPH. DPDase also catalyzed the exchange of tritium in [4S-3H,4R-1H]NADP3H with solvent protons in the absence of uracil. DPDase inactivated by 5-ethynyluracil, which covalently modifies the enzyme at the uracil binding site, catalyzed the exchange reaction at the same rate (1 s-1) as native enzyme. Thus, the interaction of NADPH with DPDase was independent of the uracil binding site. Because DPDase catalyzed the exchange of deuterium in [4S-2H,4R-1H]NADP2H with solvent protons with a rate constant of 5.4 s-1, which was significantly larger than that for tritium, the analogous rate constant for exchange of the 4-hydrogen in NADPH must be significantly larger than 5 s-1. Consequently, intermediates on the exchange pathway were kinetically competent to participate in the reduction of uracil by NADPH (kcat = 1.6 s-1). Rate constants for reduction of DPDase by NADPH and 5,6-dihydrouracil were several orders of magnitude greater than kcat. The rate constants for dissociation of E.NADP+ (15 s-1) and for dissociation of E.5,6-dihydrouracil (> 250 s-1) were also greater than kcat. These results supported a random rapid-equilibrium kinetic mechanism and suggested kcat was an internal electron transfer between enzymic prosthetic groups.
Porter
D J
DJ
Division of Experimental Therapy, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
Spector
T
T
D000818:Animals; D002417:Cattle; D003903:Deuterium; D042943:Dihydrouracil Dehydrogenase (NADP); D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008433:Mathematics; D008962:Models, Theoretical; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D011485:Protein Binding; D011865:Radioisotope Dilution Technique; D013050:Spectrometry, Fluorescence; D014316:Tritium; D014498:Uracil / Q000378:metabolism*
D014316:Tritium; D009249:NADP; D014498:Uracil; D003903:Deuterium; D010088:Oxidoreductases; D042943:Dihydrouracil Dehydrogenase (NADP)