Vitamin K epoxide reductases (VKOR) 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 reveal 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.

中文翻译：

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细胞环境中维生素K环氧化物还原的催化机制


维生素 K 环氧化物还原酶 (VKOR) 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。先前的化学模型假设人类 VKOR (hVKOR) 的催化作用是从完全还原的活性位点开始的。然而，这种状态仅构成一小部分细胞（5.6%）。因此，在细胞环境中进行 hVKOR 催化的机制仍然很大程度上未知。在这里，我们使用定量质谱 (MS) 和电泳迁移率分析表明 KO 可能与部分氧化状态下模仿 hVKOR 的半胱氨酸突变体形成共价复合物。这种潜在反应中间体的捕获表明部分氧化态在细胞中具有催化活性。为了研究这种活性，我们分析了 hVKOR 的细胞活性和细胞半胱氨酸状态之间的相关性。我们发现部分氧化的 hVKOR 的活性比活性位点完全还原的 hVKOR 低得多。尽管细胞中部分氧化的 hVKOR 多于完全还原的 hVKOR，但这两种反应状态对整体 hVKOR 活性的贡献大致相同，并且 hVKOR 催化可以从这两种状态中的任何一种开始。总体而言，MS定量和生化分析的结合揭示了这种整合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（最常用的处方药物之一）如何抑制 hVKOR。