Mechanism-based inactivation of human dihydropyrimidine dehydrogenase by (E)-5-(2-bromovinyl)uracil in the presence of NADPH

Abstract

Purified recombinant human dihydropyrimidine dehydrogenase (hDPD) was incubated with (14)C-labeled (E)-5-(2-bromovinyl)uracil ([(14)C]BVU) in the presence of NADPH to investigate a possible mechanism in the 18 patient deaths caused by interactions of 5-fluorouracil prodrugs with the new oral antiviral drug, sorivudine. BVU is formed from sorivudine by gut flora and absorbed through intestinal membrane. hDPD, a rate-limiting enzyme for the catabolism of 5-fluorouracil and endogenous pyrimidines in the human, was NADPH dependently radiolabeled and inactivated by [(14)C]BVU. Two radioactive tryptic fragments, I and II, isolated from radiolabeled hDPD were found by complete amino acid sequencing to originate from a common regional amino acid sequence located at positions 656 (Lys) to 678 (Arg) for I and positions 657 (Ser) to 678 (Arg) for II. However, only Cys(671), which should be present in the peptides, was not identified by amino acid sequencing. Mass spectrometric analysis of the tryptic fragments indicated that the sulfhydryl group of Cys(671) in the hDPD was modified with 5, 6-dihydro-5-(2-bromoethylydenyl)uracil (BEDU), a putative allyl bromide type of reactive molecule, to form a sulfide bond with loss of hydrogen bromide. The Cys(671) sulfide bearing the debrominated BEDU had a 5,6-dihydrouracil ring highly strained by the exocyclic double bond at the 5-position, so that it underwent facile hydrolytic ring fission with alkali and heated acid treatments. A new proposal is also made for the amino acid sequence of the pyrimidine-binding domain, including Cys(671), of DPD in the human and other species.