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Introduction
Biological Role
(VKOR) is a reducing enzyme composed of 4-helices that spans the endoplasmic reticulum as a transmembrane protein
[3]. Its enzymatic role is reducing (KO) to Vitamin K Hydroquinone (KH2)
[4] (Figure 1). The mechanism first occurs through the binding KO and using two cysteine residues to reduce KO into
Vitamin K. Then, a second pair of cysteine residues will reduce Vitamin K into the final product, KH2 (Figure 1). One of VKORs primary roles is to assist in blood coagulation through this KH2 regeneration mechanism.
Figure 1. Mechanism of KO oxidation into KH2.
With Vitamin K as a cofactor, the
γ-carboxylase enzyme will enact post-translational modification on KH2, oxidizing it back to KO. The oxidation of KH2 by γ-carboxylase is coupled with the carboxylation of a glutamate residue to form γ-carboxyglutamate. The coupling of this oxidation and carboxylation will activate several clotting factors in the coagulation cascade.
Author's Notes
Structural characterization of VKOR has been difficult due to its in vitro instability. Recently, a series of atomic structures have been determined utilizing anticoagulant stabilization and VKOR-like homologs. Crystal structures of VKOR were captured with a bound substrate (KO) or vitamin K antagonist (VKA) (PDB Codes: Table 1)[5]. VKA substrates utilized were anticoagulants, namely Warfarin, Brodifacoum, Phenindione, and Chlorophacinone. Second, VKOR-like homologs were utilized to aid in structure classification. Homologs refer to specific cysteine residues that have been mutated to serine to facilitate capturing a stable conformation state. Homologs were mainly isolated from human VKOR with some isolated from the pufferfish Takifugu rubripes. Furthermore, all of the structures used have been processed to remove a beta barrel at the south end of VKOR that served no purpose in function of the enzyme. This also allowed for the residue numbering to be reassigned and more closely replicate the human VKOR.
Structural Highlights
VKOR has many key structural components that allow it to maintain proper functionality and catalytic abilities. The main part of the enzyme that contains the active site is a binding pocket where main catalytic activity occurs. The VKOR binding pocket provides specific substrate binding via highly conserved residues that recognize the target substrates. The pocket works in conjunction with the cap domain. The cap domain is a helical component of VKOR that facilitates conformational transitions from the to the once a substrate binds. Interactions between the cap domain, binding pocket, and the bound protein are critical to achieve full activation of Vitamin K. Another necessary part of the structure is the anchor. The anchor serves as a way to hold VKOR in the proper orientation within the cell membrane such that all enzymatic components are in the correct proximity for substrate binding and catalysis. Vital to the VKOR structure and these components are two disulfide bridges. The first appears slightly above the binding pocket between C132 and C135. The second occurs within the cap domain between C43 and C51. These cysteines are catalytic residues that also aid in the transition of VKOR from the open conformation to the closed conformation and the reduction of KO.
Active Site
Within the four transmembrane helices lies the . The binding pocket is comprised of a containing , N80 and Y139, that interact with substrates. The hydrophobic pocket provides specificity to the region while the hydrophilic residues hydrogen bond to the substrate, providing recognition and increasing specificity. The above the binding pocket provides stabilization when a substrate is bound. This bridge provides increased stability for the binding site as it interacts with and binds substrates or inhibitors. The hydrophilic residues provide when interacting with substrates for specificity and recognition. Upon binding, VKOR will transition into the closed conformation allowing the catalytic mechanism to commence.
Cap Domain
Anchor
Function: Method of Coagulation
Brief Overview
The overall mechanism works to convert Vitamin K epoxide to an activated form of Vitamin K hydroquinone, as noted in Figure 1. The substrate will bind VKOR at the binding pocket in the and induce the . Transition from open to closed conformation occurs with the oxidation of the C43-C51 disulfide bridge. Here, VKOR will utilize the second pair of catalytic cysteines, C132 and C135, to reduce KO into Vitamin K and Vitamin K into KH2. KH2 will be released from the binding fully activated and ready for use in the body. VKOR will reset, returning to the open conformation again, prepared for another substrate to bind.
Catalytic Mechanism
The catalytic mechanism of VKOR is a critical part of its overall function in the body. Highly regulated enzymatic activity through the reactivity of catalytic cysteines allows VKOR to properly activate Vitamin K for its use in the body. The enzyme begins in , where it's in the open conformation with the cap domain open to allow in a substrate to bind to the active site. Once a substrate binds, the cap domain is initiated into the closed conformation. VKOR is now in . To further stabilize the closed conformation with the substrate bound, the cap domain helps initiate a catalytic reaction of cysteines to break the disulfide bridge that was stabilizing stage 1. Free cysteines are now available that provide strong stabilization of the closed conformation through interactions with the cap domain and the bound substrate. This puts the enzyme in , where the catalytic free cysteines react to form a new disulfide bridge, releasing the activated substrate into the blood stream to promote anticoagulation. With two stable disulfide bridges and VKOR unbound, the enzyme is now in its final, unreactive . VKOR must undergo conformational changes to return to Stage 1 and restart the catalytic process to activate Vitamin K again.
Disease and Treatment
Afflictions
Inhibition
The most inexpensive and common way to treat blood clotting is through the VKOR inhibitor, . Warfarin is able to do so by outcompeting KO. It will enter the binding pocket of VKOR, creating strong hydrogen bonds with the active site.
Mutations
Some key that can be detrimental to the VKOR structure are mutations of the . The two main residues, N80 and Y139, can be mutated to A80 and F139 creating a decrease in recognition and stabilization.
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