Sandbox Reserved 1724

This Sandbox is Reserved from February 28 through September 1, 2022 for use in the course CH462 Biochemistry II taught by R. Jeremy Johnson at the Butler University, Indianapolis, USA. This reservation includes Sandbox Reserved 1700 through Sandbox Reserved 1729.

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Vitamin K Epoxide Reductase

spinqualitylabelsall models Overall Structure of Vitamin K Epoxide Reductase Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Introduction Vitamin K epoxide reductase (VKOR) is the enzyme responsible for regenerating vitamin K from vitamin K epoxide to support blood coagulation. Figure 1. Vitamin K Cycle Figure 2. VKOR with Barrel Domain Vitamin K Cycle Vitamin K is essential for blood clotting in the body. The fully reduced form, KH2, allows the gamma carboxylation of blood clotting cofactors and is turned into the epoxide form in the process. Vitamin K epoxide reductase, abbreviated VKOR, turns the epoxide back to the fully reduced form so the reduced form can be used again. This transformation happens in two steps including converting the epoxide to the partially oxidized Vitamin K quinone then converting the quinone to the fully reduced hydroquinone (KH2). Structural Overview VKOR consists of four transmembrane helices embedded in the endoplasmic reticulum membrane. The files on the RCSB Protein Data Bank include a barrel domain that is not pertinent to the function of VKOR. The images presented here have been edited to remove the barrel domain and renumbered to correspond with the article by Liu. . The original image with the barrel domain in context is shown in Figure 2. Helices one and two are connected by the beta hairpin region which contains two of the active cysteines, C43 and C51. VKOR also has a cap domain covering the active site, made up of an anchor, loop, and helix. Catalytic Cycle Catalytic Cysteines Figure 3. Catalytic Cycle of VKOR VKOR's luminal domain is represented by a the pink semicircle and the transmembrane domain is represented by the gray cylinder. State I displays the fully oxidized VKOR in an open conformation. State II is a partially oxidized VKOR with free cysteines 43 and 135. State III is cysteine 135 interacting with KOH or KH to induce a closed conformation. State IV shows cysteine 132 attacking cysteine 135-KOH or KH to generate K. The catalytic cycle of VKOR includes transitions from open to closed conformations by means of disulfide bridge-induced conformational changes (Figure 3). Open conformations of VKOR exist when there is no ligand within the binding pocket. Closed conformations exist when some substrate exists within the binding pocket of VKOR. The substituent cysteines (Fig 3, step I) act as reducing agents for the substrate, which can be either Vitamin K epoxide (KO) or partially reduced Vitamin K. The first step of the catalytic cycle (Figure 3) is the wild type open conformation, step I. This step is characterized by an open cap domain with disulfide bonds between cysteines 43 and 51 and between cysteines 132 and 135 . The second step of the catalytic cycle is a partially oxidized open conformation, step II. This step is characterized by a disulfide bond between the luminal and transmembrane domain (Fig 3, step II). The transmembrane domain contains a free Cys135 and the luminal domain contains a free Cys43 . Step II is labeled as open because no ligand exists within its binding pocket despite the disulfide bridge that connects the luminal and transmembrane domains. The next step of the cycle, step III, is also a closed structure with an intact disulfide bond between Cys51 and Cys132. Cys135 is not involved in a disulfide bridge and assists with substrate binding by forming a stable adduct with KOH or K. This binding induces the closed conformation and uses Cys43 in the luminal membrane for electron transfer . Step IV of the catalytic cycle is the last closed conformation. The Cys51-Cys132 bond is broken as Cys43 bonds with Cys51, recreating the disulfide bridge pattern of the open state. Cys132 is then free to bond with Cys135, releasing the product that was bound to the Cys135. Overall the catalytic cycle of VKOR is dependent on open and closed conformational changes of the protein and ultimately is used to generate vitamin K from vitamin K epoxide . Warfarin Dependence on Catalytic Cysteines The anticoagulant warfarin works by inhibiting VKOR (See "Medical Relevance"). Warfarin binding also depends on the catalytic cysteines. Warfarin is able to bind to the fully oxidized open form of VKOR as shown in step I. Once Warfarin binds, VKOR is considered to be in a closed conformation since the substrate cannot enter, despite the lack of disulfide bridge changes. Warfarin can also bind to the partially oxidized form of VKOR as shown in step II. Key Substrate Binding Residues VKOR uses two catalytic amino acids, tyrosine 139 and asparagine 80, to stabilize binding to all forms of vitamin K and vitamin K antagonists, such as Warfarin, in the binding pocket. Tyr139 and Asn80 hydrogen bond to carbonyl groups on both structures and stabilizes them within the binding pocket . Hydrophobic Interactions Other than the two previously mentioned hydrogen bonds (Tyr139 and Asn80), vitamin K and VKOR antagonists are bound via hydrophobic interactions within the binding pocket of VKOR. Hydrophobic residues of VKOR such as Phe80, Phe87, and Tyr88, form a hydrophobic tunnel within the binding pocket . Medical Relevance Warfarin Warfarin is the most widely prescribed oral anticoagulant and targets blood clotting via inhibition of vitamin K epoxide reductase. The FDA approved uses for cardiac conditions (myocardial infarction, atrial fibrillation) as well as for deep vein thrombosis and pulmonary embolism. Due to the inhibition of the normal blood clotting cycle, patients taking warfarin are at risk for hemorrhage which can occur anywhere in the body. Warfarin is a structural mimic of Vitamin K and acts as a competitive inhibitor. There are around 30 known missense mutations that lead to warfarin resistance in patients, but these mutations do not affect Vitamin K binding for reasons which are not yet fully understood. Such patients require higher doses of warfarin to reach therapeutic level or require a different anticoagulant drug. Superwarfarins Figure 4. Warfarin and Brodifacoum More potent warfarin derivatives, called superwarfarins, are used as rodenticides. Superwarfarins have bulkier side chains that allow them to stay bound to VKOR for long periods of time, causing prolonged and uncontrolled bleeding. The duration of one superwarfarin, brodifacoum, has been reported as 15-30 days vs. the clinical warfarin duration of 2-5 days.

Student Contributors

Izabella Jordan, Emma Varness