Preusch P C, Smalley D M
Department of Chemistry, University of Akron, OH 44325.
Free Radic Res Commun. 1990;8(4-6):401-15. doi: 10.3109/10715769009053374.
The chemical and enzymatic pathways of vitamin K1 epoxide and quinone reduction have been investigated. The reduction of the epoxide by thiols is known to involve a thiol-adduct and a hydroxy vitamin K enolate intermediate which eliminates water to yield the quinone. Sodium borohydride treatment resulted in carbonyl reduction generating relatively stable compounds that did not proceed to quinone in the presence of base. NAD(P)H:quinone oxidoreductase (DT-diaphorase, E.C. 1.6.99.2) reduction of vitamin K to the hydroquinone was a significant process in intact microsomes, but 1/5th the rate of the dithiothreitol (DTT)-dependent reduction. No evidence was found for DT-diaphorase catalyzed reduction of vitamin K1 epoxide, nor was it capable of mediating transfer of electrons from NADH to the microsomal epoxide reducing enzyme. Purified diaphorase reduced detergent- solubilized vitamin K1 10(-5) as rapidly as it reduced dichlorophenylindophenol (DCPIP). Reduction of 10 microM vitamin K1 by 200 microM NADH was not inhibited by 10 microM dicoumarol, whereas DCPIP reduction was fully inhibited. In contrast to vitamin K3 (menadione), vitamin K1 (phylloquinone) did not stimulate microsomal NADPH consumption in the presence or absence of dicoumarol. DTT-dependent vitamin K epoxide reduction and vitamin K reduction were shown to be mutually inhibitory reactions, suggesting that both occur at the same enzymatic site. On this basis, a mechanism for reduction of the quinone by thiols is proposed. Both the DTT-dependent reduction of vitamin K1 epoxide and quinone, and the reduction of DCPIP by purified DT-diaphorase were inhibited by dicoumarol, warfarin, lapachol, and sulphaquinoxaline.
维生素K1环氧化物和醌还原的化学及酶促途径已得到研究。已知硫醇对环氧化物的还原涉及硫醇加合物和羟基维生素K烯醇化物中间体，该中间体消除水生成醌。硼氢化钠处理导致羰基还原，生成相对稳定的化合物，在有碱存在的情况下不会进一步生成醌。在完整的微粒体中，NAD(P)H：醌氧化还原酶（DT-黄递酶，E.C. 1.6.99.2）将维生素K还原为氢醌是一个重要过程，但速率仅为二硫苏糖醇（DTT）依赖性还原速率的五分之一。未发现有证据表明DT-黄递酶催化维生素K1环氧化物的还原，它也不能介导电子从NADH转移至微粒体环氧化物还原酶。纯化的黄递酶还原去污剂溶解的维生素K1的速度与还原二氯酚靛酚（DCPIP）的速度一样快，为10^(-5) 。200 microM NADH对10 microM维生素K1的还原不受10 microM双香豆素的抑制，而DCPIP的还原则完全受抑制。与维生素K3（甲萘醌）不同，无论有无双香豆素存在，维生素K1（叶绿醌）均不刺激微粒体NADPH的消耗。DTT依赖性维生素K环氧化物还原和维生素K还原被证明是相互抑制的反应，这表明两者发生在同一酶位点。在此基础上，提出了硫醇还原醌的机制。双香豆素、华法林、拉帕醇和磺胺喹恶啉均抑制DTT依赖性维生素K1环氧化物和醌的还原以及纯化的DT-黄递酶对DCPIP的还原。
