The catalytic mechanism of vitamin K epoxide reduction in a cellular environment.
The catalytic mechanism of vitamin K epoxide reduction in a cellular environment.
复制标题
细胞环境中维生素 K 环氧化物还原的催化机制。
DOI：
10.1074/jbc.ra120.015401
复制
发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Li W
中科院分区：
文献类型：
--
作者：
Shen G;Cui W;Cao Q;Gao M;Liu H;Su G;Gross ML;Li W
维生素K环氧化物还原酶（VKORs）是完整膜硫醇氧化还原酶的一个主要家族。在人类中，VKOR通过维生素K循环维持血液凝固和骨矿化。以前的化学模型假设人类VKOR （hVKOR）的催化作用从一个完全还原的活性位点开始。然而，这种状态只占细胞的一小部分（5.6%）。因此，hVKOR催化在细胞环境中进行的机制在很大程度上仍然未知。在这里，我们使用定量质谱（MS）和电泳迁移率分析表明，KO可能与半胱氨酸突变体在部分氧化状态下形成共价复合物，模拟hVKOR。捕获这种潜在的反应中间体表明部分氧化状态在细胞中具有催化活性。为了研究这种活性，我们分析了细胞活性与hVKOR细胞半胱氨酸状态之间的相关性。我们发现部分氧化的hVKOR的活性明显低于活性位点完全还原的hVKOR。虽然细胞中部分氧化的hVKOR多于完全还原的hVKOR，但这两种反应状态对hVKOR的总体活性贡献大致相同，并且hVKOR催化可以从这两种状态中任一状态启动。综上所述，质谱定量和生化分析相结合揭示了这种整体膜酶在细胞环境中的催化机制。此外，这些结果暗示了华法林（最常用的处方药之一）是如何抑制hVKOR的。
Vitamin K epoxide reductases (VKORs) constitute a major family of integral membrane thiol oxidoreductases. In humans, VKOR sustains blood coagulation and bone mineralization through the vitamin K cycle. Previous chemical models assumed that the catalysis of human VKOR (hVKOR) starts from a fully reduced active site. This state, however, constitutes only a minor cellular fraction (5.6%). Thus, the mechanism whereby hVKOR catalysis is carried out in the cellular environment remains largely unknown. Here we use quantitative mass spectrometry (MS) and electrophoretic mobility analyses to show that KO likely forms a covalent complex with a cysteine mutant mimicking hVKOR in a partially oxidized state. Trapping of this potential reaction intermediate suggests that the partially oxidized state is catalytically active in cells. To investigate this activity, we analyze the correlation between the cellular activity and the cellular cysteine status of hVKOR. We find that the partially oxidized hVKOR has considerably lower activity than hVKOR with a fully reduced active site. Although there are more partially oxidized hVKOR than fully reduced hVKOR in cells, these two reactive states contribute about equally to the overall hVKOR activity, and hVKOR catalysis can initiate from either of these states. Overall, the combination of MS quantification and biochemical analyses reveals the catalytic mechanism of this integral membrane enzyme in a cellular environment. Furthermore, these results implicate how hVKOR is inhibited by warfarin, one of the most commonly prescribed drugs.