Nishiyama T, Ogura K, Okuda H, Suda K, Kato A, Watabe T
Department of Drug Metabolism and Molecular Toxicology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan.
Mol Pharmacol. 2000 May;57(5):899-905.
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.
将纯化的重组人二氢嘧啶脱氢酶（hDPD）与在NADPH存在下的（14）C标记的（E）-5-（2-溴乙烯基）尿嘧啶（[（14）C]BVU）一起孵育，以研究5-氟尿嘧啶前药与新型口服抗病毒药物索立夫定相互作用导致18例患者死亡的可能机制。BVU由肠道菌群从索立夫定形成并通过肠膜吸收。hDPD是人体内5-氟尿嘧啶和内源性嘧啶分解代谢的限速酶，被[（14）C]BVU以NADPH依赖的方式进行放射性标记并失活。通过完整的氨基酸测序发现，从放射性标记的hDPD中分离出的两个放射性胰蛋白酶片段I和II，分别源自位于656位（赖氨酸）至678位（精氨酸）的共同区域氨基酸序列（片段I）和位于657位（丝氨酸）至678位（精氨酸）的共同区域氨基酸序列（片段II）。然而，通过氨基酸测序仅未鉴定出应存在于肽中的半胱氨酸（671）。对胰蛋白酶片段的质谱分析表明，hDPD中半胱氨酸（671）的巯基被5,6-二氢-5-（2-溴乙基）尿嘧啶（BEDU，一种假定的烯丙基溴类型的反应性分子）修饰，形成了一个失去溴化氢的硫醚键。带有脱溴BEDU的半胱氨酸（671）硫醚具有一个5,6-二氢尿嘧啶环，该环因5位的环外双键而高度紧张，因此在碱处理和加热酸处理下容易发生水解环开裂。还对人和其他物种中DPD的嘧啶结合结构域（包括半胱氨酸（671））的氨基酸序列提出了新的建议。
