Sandbox Reserved 1725

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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 The three states of Vitamin K are shown along with important enzymes, including VKOR. Vitamin K Cycle Vitamin K is essential for blood clotting in the body. The fully reduced form, KH2, is used by gamma-glutamyl carboxylase to carboxylate protein-bound glutamate residues in blood clotting cofactor precursors . After carboxylation, the clotting cofactors (such as prothrombin) can bind to calcium and can proceed to the coagulation cascade . During this process, KH2 becomes oxidized to Vitamin K epoxide, or KO . 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: 1) converting the epoxide to the partially oxidized Vitamin K quinone and 2) converting the quinone to the fully reduced hydroquinone (KH2) (Figure 1) . Figure 2. VKOR in Membrane Transmembrane helices are teal. The tan sections are the loops (loop 1, beta hairpin, cap loop, 3-4 loop). The cap helix is light blue and the anchor is dark blue. Structural Overview VKOR consists of four transmembrane helices embedded in the endoplasmic reticulum membrane. Helices one and two are connected by Loop 1 and the beta hairpin region which contains two of the active cysteines, C43 and C51; these cysteines, along with C132 and C135, are essential for reduction and structural changes discussed in the next section. VKOR also has a cap domain consisting of a helix, loop, and anchor. The anchor serves to attach the cap domain to the ER membrane for stabilization. The loop helps stabilize one of the catalytic amino acids, Asn80. The helix is involved in stabilization of certain disulfide bonds and structural changes as part of the catalytic cycle discussed below. Author's Note In order to deduce VKOR structures, Liu and colleagues used various strategies worthy of note. They incorporated a barrel-like domain that is not part of the catalytic VKOR protein to help with stability during experimentation. In some conformations, a catalytic cysteine was mutated to serine to force the reaction to stop with VKOR in that certain conformation. Also, due to stability issues, some of Liu's experiments used human VKOR and others used VKOR of Takifugu rubripes which is similar to humans. The barrel domain has been removed here and the amino acids renumbered to more closely match the published numbering in the referenced article . Active Site VKOR uses two catalytic amino acids, tyrosine 139 and asparagine 80 to stabilize vitamin K in the binding pocket. Tyr139 and Asn80 hydrogen bond to carbonyl groups on both structures and stabilizes them within the binding pocket . Other than the two previously mentioned hydrogen bonds (Tyr139 and Asn80), vitamin K is also bound via hydrophobic interactions within the binding pocket of VKOR. Hydrophobic residues of VKOR such as Phe83, Phe87, Tyr88, form a hydrophobic tunnel within the binding pocket . Catalytic Cycle Catalytic Cysteines 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. 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. Step I and II represent open conformations of VKOR and steps III and IV represent closed conformations. 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 . 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 that occupies the VKOR binding site, acting as a competitive inhibitor. Warfarin mimics vitamin K by binding to the same substrate binding residues(Asn80 and Tyr139) in the active site. Warfarin also shares the same hydrophobic interactions within the binding site that KOH experiences (Phe83, Phe87, and Tyr88). Warfarin binding also depends on the VKOR catalytic cysteines. Warfarin is able to bind to the fully oxidized open form of VKOR as shown in step I of the catalytic cycle. 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 of the catalytic cycle. 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. Vitamin K, Warfarin, and Brodifacoum Above is a comparison of the 2D structures of VKOR's natural substrate, the blood thinner warfarin, and the superwarfarin brodifacoum. More potent warfarin derivatives, called superwarfarins, are used as rodenticides. The duration of one superwarfarin, brodifacoum, has been reported as 15-30 days vs. the clinical warfarin duration of 2-5 days. Superwarfarins have bulkier side chains that allow them to stay bound to VKOR for long periods of time, causing prolonged and uncontrolled bleeding. The enhanced binding is due to interactions between the bulky side chains and the hydrophobic residues of VKOR's binding pocket, brodifacoum.

Student Contributors

Izabella Jordan, Emma Varness