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Introduction

Biological Role

Figure 1. Mechanism of KO oxidation into KH2.

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 , 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.

Enzymatic Mechanism

The catalytic mechanism of VKOR is highly regulated and use to activate Vitamin K necessary for blood coagulation. Figure 3 highlights these reactions that allow the substrate to be catalyzed to its active form through a series of 4 stages. The enzyme begins in in the open conformation with the cap domain open to allow substrate binding. Once a substrate binds, the cap domain transitions to the closed conformation when the C51-C132 disulfide bridge is attacked by reactive C43 located within the cap domain. This reaction forms a new disulfide bridge between C43 and C51 that pulls the cap domain over the binding pocket with the substrate bound to stabilize the closed conformation of VKOR. VKOR is now in . 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 a free C135 is purposed to interact with the substrate within the binding pocket to stabilize it during activation. The catalytic free C132 located between the cap domain and helical tunnel is very reactive and will attack this C135 to break that interaction with the substrate and release the activated Vitamin K product into the blood stream to promote coagulation. Two very stable disulfide bridges between C43-C41 and C132-C135 are now present and VKOR is unbound, so the enzyme is in its final, unreactive . VKOR must undergo conformational changes to return to Stage 1 and reactivate its catalytic cysteines so that another molecule of Vitamin K can bind and be activated.

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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