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Characteristics of recombinant W501S mutated human γ‐glutamyl carboxylase
Author(s) -
Soute B. A. M.,
Jin DY.,
Spronk H. M. H.,
Mutucumarana V. P.,
Lin PJ.,
Hackeng T. M.,
Stafford D. W.,
Vermeer C.
Publication year - 2004
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2004.00686.x
Subject(s) - pyruvate carboxylase , protein precursor , recombinant dna , carboxylation , mutant , chemistry , enzyme , mutation , biochemistry , vitamin , wild type , microbiology and biotechnology , biology , gene , catalysis
Summary. A mutation (W501S) in the vitamin K‐dependent γ‐glutamyl carboxylase (VKC) that leads to a congenital bleeding disorder was recently discovered in two patients. To characterize the enzyme defect, recombinant VKC‐W501S was expressed in and purified from insect cells. The major effect of the mutation appears to be to decrease the affinity of the carboxylase for the propeptide of its substrates. This observation agrees with recent data that place part of the propeptide binding site within residues 495–513 of VKC. Additionally, we demonstrate that the affinity between descarboxy osteocalcin (d‐OC) and VKC remains unaffected by the W501S mutation. This confirms earlier data that the high‐affinity site for d‐OC is not located on the propeptide binding domain of VKC. Two properties of the enzyme suggest an explanation for the observation that vitamin K supplementation ameliorates the effects of the mutation: (i) since full carboxylation requires the propeptide to remain bound to the enzyme sufficiently long for full carboxylation, a reduced affinity can cause its premature release before carboxylation is complete; (ii) propeptide binding results in a decrease of the K M for vitamin K hydroquinone in wild‐type, but not in mutant carboxylase, resulting in increased vitamin K requirement of affected subjects.