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

被引:83
作者
Rost, S
Fregin, A
Hünerberg, M
Bevans, CG
Müller, CR
Oldenburg, J
机构
[1] Univ Clin Bonn, Inst Expt Haematol & Transfus Med, D-53127 Bonn, Germany
[2] Univ Wurzburg, Bioctr, Inst Human Genet, Wurzburg, Germany
[3] Max Planck Inst Biophys, Dept Biol Struct, D-6000 Frankfurt, Germany
[4] Inst Transfus Med & Immunohaematol, DRK Blood Donor Serv Baden Wurttemberg Hessen, Frankfurt, Germany
关键词
vitamin K; VKOR; warfarin; CXXC motif; mutagenesis;
D O I
10.1160/TH05-02-0082
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
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 (Cys 16, Cys43, Cys51, Cys85, Cys96, Cys 132, Cys 135), the highly conserved residue Ser/Thr57, and Arg98, known to cause VKCFD2 in humans. Our results support the hypothesis that the CI32-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.
引用
收藏
页码:780 / 786
页数:7
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