A fluorescent method to determine vitamin K-dependent gamma-glutamyl carboxylase activity.

The gamma (γ)-glutamyl carboxylase is a key enzyme in vitamin K-dependent carboxylation of proteins involved in hemostasis and inflammation. It is an endoplasmic enzyme posttranslationally converting glutamic acid residues into γ-carboxyglutamic acid residues in proteins. The activity of tissue derived γ-glutamyl carboxylase is commonly assayed by incorporation of H¹⁴CO₃⁻ into synthetic peptides and subsequent quantification using liquid scintillation counting.

Effect of vitamin K-dependent protein precursor propeptide, vitamin K hydroquinone, and glutamate substrate binding on the structure and function of {gamma}-glutamyl carboxylase.

The γ-glutamyl carboxylase utilizes four substrates to catalyze carboxylation of certain glutamic acid residues in vitamin K-dependent proteins. How the enzyme brings the substrates together to promote catalysis is an important question in understanding the structure and function of this enzyme. The propeptide is the primary binding site of the vitamin K-dependent proteins to carboxylase.

Two-dimensional crystallization of human vitamin K-dependent gamma-glutamyl carboxylase.

Planar-tubular two-dimensional (2D) crystals of human vitamin K-dependent gamma-glutamyl carboxylase grow in the presence of dimyristoyl phosphatidylcholine (DMPC). Surprisingly, these crystals form below the phase transition temperature of DMPC and at the unusually low molar lipid-to-protein (LPR) ratio of 1, while 2D crystals are conventionally grown above the phase transition temperature of the reconstituting lipid and significantly higher LPRs. The crystals are up to 0.

A conserved region of human vitamin K-dependent carboxylase between residues 393 and 404 is important for its interaction with the glutamate substrate.

Certain individuals with combined deficiencies of vitamin K-dependent proteins have a mutation, L394R, in their gamma-glutamyl carboxylase causing impaired glutamate binding. The sequence surrounding Leu394 is similar in all known carboxylases, suggesting that the region is functionally important. To test this hypothesis we made the following mutant enzymes: W390A, Y395A, S398A, W399A, and H404A.

Determination of disulfide bond assignment of human vitamin K-dependent gamma-glutamyl carboxylase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Vitamin K-dependent gamma-glutamyl carboxylase is a 758 amino acid integral membrane glycoprotein that catalyzes the post-translational conversion of certain protein glutamate residues to gamma-carboxyglutamate. Carboxylase has ten cysteine residues, but their form (sulfhydryl or disulfide) is largely unknown. Pudota et al.