Site-directed mutagenesis of coumarin-type anticoagulant-sensitive VKORC1: evidence that highly conserved amino acids define structural requirements for enzymatic activity and inhibition by warfarin

Thromb Haemost. 2005 Oct;94(4):780-6. doi: 10.1160/TH05-02-0082.


Coumarin and homologous compounds are the most widely used anticoagulant drugs worldwide. They function as antagonists of vitamin K, an essential cofactor for the posttranslational gamma-glutamyl carboxylation of the so-called vitamin K-dependent proteins. As vitamin K hydroquinone is converted to vitamin K epoxide (VKO) in every carboxylation step, the epoxide has to be recycled to the reduced form by the vitamin K epoxide reductase complex (VKOR). Recently, a single coumarin-sensitive protein of the putative VKOR enzyme complex was identified in humans (vitamin K epoxide reductase complex subunit 1, VKORC1). Mutations in VKORC1 result in two different phenotypes: warfarin resistance (WR) and multiple coagulation factor deficiency type 2 (VKCFD2). Here,we report on the expression of site-directed VKORC1 mutants, addressing possible structural and functional roles of all seven cysteine residues (Cys16, Cys43, Cys51, Cys85, Cys96, Cys132, Cys135), the highly conserved residue Ser/Thr57, and Arg98, known to cause VKCFD2 in humans. Our results support the hypothesis that the C132-X-X-C135 motif in VKORC1 comprises part of the redox active site that catalyzes VKO reduction and also suggest a crucial role for the hydrophobic Thr-Tyr-Ala motif in coumarin binding. Furthermore, our results support the concept that different structural components of VKORC1 define the binding sites for vitamin K epoxide and coumarin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Anticoagulants / metabolism
  • Anticoagulants / pharmacology*
  • Binding Sites / genetics
  • Cell Line
  • Coumarins / metabolism
  • Coumarins / pharmacology*
  • Disulfides / metabolism
  • Drug Resistance / genetics*
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Kidney / cytology
  • Mixed Function Oxygenases / chemistry
  • Mixed Function Oxygenases / genetics*
  • Mixed Function Oxygenases / metabolism
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Vitamin K 1 / analogs & derivatives
  • Vitamin K 1 / metabolism
  • Vitamin K Epoxide Reductases


  • Anticoagulants
  • Coumarins
  • Disulfides
  • vitamin K1 oxide
  • Vitamin K 1
  • coumarin
  • Mixed Function Oxygenases
  • VKORC1 protein, human
  • Vitamin K Epoxide Reductases