Study On The Mechanism Of Human Vitamin K Epoxide Reductase Catalyzing Vitamin K Epoxide

Human vitamin K epoxide reductase(h VKOR)is an integral membrane protein located in the endoplasmic reticulum.It catalyzes the rate-limiting step in the vitamin K cycle and participates in the post-translational modification of vitamin K-dependent proteins.Warfarin,a commonly used anticoagulant in clinical practice,specifically targets h VKOR to block the vitamin K cycle,and the γ-carboxylation of coagulation factors synthesized by the liver is inhibited to produce an anticoagulant effect.At present,the mechanism of h VKOR catalyzing KO is proposed based on quantum chemistry theory,but the specific catalytic mechanism in the cellular environment is not clear.Therefore,it is of great theoretical significance to deeply study the catalytic mechanism of h VKOR to KO under physiological conditions,and it is also helpful to understand the inhibition mechanism of warfarin.Objectives:By constructing h VKOR wild-type and mutant stable cell lines,gel migration experiments,quantitative mass spectrometry,and molecular simulation were used to capture the electron transfer intermediates formed by VKOR in the process of catalyzing KO,and further reveal the catalytic mechanism of VKOR catalyzing KO in the cell environment。Methods:1.Using pc DNA5 / FRT as a vector,a rapid cloning method was used to construct recombinant plasmids expressing wild-type h VKOR and cysteine mutants,and the constructions were verified by sequencing.2.293T-Rex cells were routinely cultured in vitro,and the successfully sequenced recombinant plasmids pc DNA5 / FRT and p OG44 were co-transfected into 293T-Rex cells at a ratio of 1:10.After 48 hours,a certain concentration of antibiotics(blasticidin and hygromycin)was added to the medium to screen stable cell lines.and the target protein was induced to express by adding doxycycline to the medium.3.A KO-induced gel migration experiment was established.The cells stably expressing wild-type h VKOR and related cysteine mutants were divided into KO treated group and untreated group,and were subjected to reduced and non-reduced SDS-PAGE electrophoresis,the effects of KO treatment on the electrophoretic migration of h VKOR mutants were detected by Western Blot detection.4.Proteins expressing wild-type h VKOR and each cysteine mutant were purified from stable cell lines,and the redox state of each cysteine was analyzed by quantitative mass spectrometry.5.Combined with the structure of bacterial ss VKOR,a computer simulation method was used to construct the complex structure model formed by the combination of h VKOR and KO and h VKOR and warfarin respectively.Results:1.Recombinant plasmids expressing wild-type h VKOR and each mutant were successfully constructed using pc DNA5 / FRT.2.293T-Rex cells were successfully used to construct a cell line stably expressing wild-type h VKOR and each mutant.3.The results of gel-shift assay induced by KO showed that after the C43 A mutant was treated with KO,a shift-band appeared in non-reduced SDS-PAGE electrophoresis.The shift-band disappeared in the reduced SDS-PAGE electrophoresis,while other mutants did not appear the shift-band phenomenon;In the C43AC135 A and C43AC135 S double mutants,the same method was used after KO induction treatment,and non-reduced SDS-PAGE electrophoresis was performed.It was found that KO-induced gel-shift in C43 A disappeared in both double mutations.4.Quantitative mass spectrometry analysis of wild-type h VKOR and each mutant showed that C135 in the C43 A mutant became oxidized after adding KO treatment,and KO treatment also induced more C16 and C85 in the C43 A mutant to be oxidized;The redox state of C135 in other mutants before and after KO treatment did not change significantly.Except for the C43 A mutation,the redox states of C16,C85,and C96 in other mutants did not change significantly before and after KO treatment.Combining the above results,we constructed a catalytic mechanism model of h VKOR catalyzing KO.5.A computer simulation method was used to successfully construct the complex structure models formed by the combination of h VKOR and KO and h VKOR and warfarin.Suggesting that KO and warfarin combine in the same pocket,and then we propose a model of warfarin to inhibit h VKOR.Conclusion:1.In the cells,h VKOR has two or three active forms,It is the state that C51-C132 forms a disulfide bond and C43 and C135 are free sulfhydryl groups,and the state with two free thiol groups of C132 and C135.The efficiency of catalyzing KO in these states may be different.2.Since there are two or three active forms of h VKOR in cells,there are several mechanisms by which h VKOR catalyzes KO to K.Intracellular h VKOR catalyzes KO is a mixed catalytic mechanism.3.Based on the mixed catalytic model of h VKOR catalyzed KO in cells,we believe that warfarin inhibits h VKOR activity through a mixed inhibition mode.