(R)-5-fluoro-5,6-dihydrouracil: kinetics of oxidation by dihydropyrimidine dehydrogenase and hydrolysis by dihydropyrimidine aminohydrolase.
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.
Porter
D J
DJ
Division of Experimental Therapy, Wellcome Research Laboratories, Research Triangle Park, NC 27709.
Harrington
J A
JA
Almond
M R
MR
Lowen
G T
GT
Spector
T
T
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
C019389:5-fluorodihydrouracil; D010088:Oxidoreductases; D042943:Dihydrouracil Dehydrogenase (NADP); D000581:Amidohydrolases; C020047:dihydropyrimidinase; D005472:Fluorouracil