The catalytic mechanism of vitamin K epoxide reduction in a cellular environment.

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.

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Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.