title: (R)-5-fluoro-5,6-dihydrouracil: kinetics of oxidation by dihydropyrimidine dehydrogenase and hydrolysis by dihydropyrimidine aminohydrolase.
abstract: The biologically active isomer of 5-fluoro-5,6-dihydrouracil [(R)-5-fluoro-5,6-dihydrouracil, R-FUH2] was synthesized to study the kinetics of its enzymatic oxidation and hydrolysis by homogeneous dihydropyrimidine dehydrogenase (DPDase) and dihydropyrimidine aminohydrolase (DPHase), respectively. DPDase catalyzed the slow oxidation of R-FUH2 at pH 8 and 37 degrees with a Km of 210 microM and a kcat of 0.026 sec-1 at a saturating concentration of NADP+. The catalytic efficiency (kcat/Km) of DPDase for R-FUH2 was 1/14th of that for 5,6-dihydrouracil (UH2). In the opposite direction, DPDase catalyzed the reduction of 5-fluorouracil (FU) with a Km of 0.70 microM and a kcat of 3 sec-1 at a saturating concentration of NADPH. Thus, DPDase catalyzed the reduction of FU 30,000-fold more efficiently than the oxidation of R-FUH2. In contrast to the slow oxidation of R-FUH2 by DPDase, R-FUH2 was hydrolyzed very efficiently by DPHase with a Km of 130 microM and a kcat of 126 sec-1. The catalytic efficiency of DPHase for the hydrolysis of R-FUH2 was approximately twice that for the hydrolysis of UH2. Because R-FUH2 is hydrolysis of R-FUH2 was approximately twice that for the hydrolysis of UH2. Because R-FUH2 is hydrolyzed considerably more efficiently than it is oxidized and because the activity of DPHase was 250- to 500-fold greater than that of DPDase in bovine and rat liver, the hydrolytic pathway should predominate in vivo.
authors.affiliation: Division of Experimental Therapy, Wellcome Research Laboratories, Research Triangle Park, NC 27709.
authors.forename: D J
authors.initials: DJ
authors.lastname: Porter
authors.forename: J A
authors.initials: JA
authors.lastname: Harrington
authors.forename: M R
authors.initials: MR
authors.lastname: Almond
authors.forename: G T
authors.initials: GT
authors.lastname: Lowen
authors.forename: T
authors.initials: T
authors.lastname: Spector
mesh_terms: D000581:Amidohydrolases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D042943:Dihydrouracil Dehydrogenase (NADP); D005472:Fluorouracil / Q000031:analogs & derivatives* / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013237:Stereoisomerism
chemical_list: C019389:5-fluorodihydrouracil; D010088:Oxidoreductases; D042943:Dihydrouracil Dehydrogenase (NADP); D000581:Amidohydrolases; C020047:dihydropyrimidinase; D005472:Fluorouracil