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Dihydropyrimidine dehydrogenase. Kinetic mechanism for reduction of uracil by NADPH.
J Biol Chem. 1993 Sep 15; 268(26):19321-7.JB

Abstract

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.

Authors+Show Affiliations

Division of Experimental Therapy, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.No affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

8366081

Citation

Porter, D J., and T Spector. "Dihydropyrimidine Dehydrogenase. Kinetic Mechanism for Reduction of Uracil By NADPH." The Journal of Biological Chemistry, vol. 268, no. 26, 1993, pp. 19321-7.
Porter DJ, Spector T. Dihydropyrimidine dehydrogenase. Kinetic mechanism for reduction of uracil by NADPH. J Biol Chem. 1993;268(26):19321-7.
Porter, D. J., & Spector, T. (1993). Dihydropyrimidine dehydrogenase. Kinetic mechanism for reduction of uracil by NADPH. The Journal of Biological Chemistry, 268(26), 19321-7.
Porter DJ, Spector T. Dihydropyrimidine Dehydrogenase. Kinetic Mechanism for Reduction of Uracil By NADPH. J Biol Chem. 1993 Sep 15;268(26):19321-7. PubMed PMID: 8366081.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Dihydropyrimidine dehydrogenase. Kinetic mechanism for reduction of uracil by NADPH. AU - Porter,D J, AU - Spector,T, PY - 1993/9/15/pubmed PY - 1993/9/15/medline PY - 1993/9/15/entrez SP - 19321 EP - 7 JF - The Journal of biological chemistry JO - J Biol Chem VL - 268 IS - 26 N2 - 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. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/8366081/Dihydropyrimidine_dehydrogenase__Kinetic_mechanism_for_reduction_of_uracil_by_NADPH_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(19)36516-0 DB - PRIME DP - Unbound Medicine ER -