(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 -FUH 2 ] 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 -FUH 2 at pH 8 and 37° with a K m of 210 μM and a k cat of 0.026 sec −1 at a saturating concentration of NADP + . The catalytic efficiency ( k cat / k m ) of DPDase for R -FUH 2 was 1 14 th of that for 5,6-dihydrouracil (UH 2 ). In the opposite direction, DPDase catalyzed the reduction of 5-fluorouracil (FU) with a K m of 0.70 μM and a k cat 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 -FUH 2 . In contrast to the slow oxidation of R -FUH 2 by DPDase, R -FUH 2 was hydrolyzed very efficiently by DPHase with a K m of 130, μM and a k cat of 126 sec −1 . The catalytic efficiency of DPHase for the hydrolysis of R -FUH 2 was approximately twice that for the hydrolysis of UH 2 . Because R -FUH 2 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 .