Investigation of the Binding Site Between the Vitamin K-Dependent Enzyme Gamma-Glutamyl Carboxylase and its Substrates

Gamma-glutamyl carboxylase is a vitamin K-dependent (VKD) integral membrane protein that converts specific glutamic acid residues (Glu) into gamma-carboxyglutamic acid residues (Gla). This post-translational modification is important in various biological functions such as blood coagulation, bone metabolism, and growth control.

Carboxylase is a 758 amino acid protein with five transmembrane domains1 that recognizes the VKD proteins through an amino-terminal highly-conserved eighteen amino acid region, termed the propeptide2. Immediately following the propeptide is the “Gla domain”. This approximately 45 amino acid residue region contains the Glu residues that are converted to Gla. In a typical VKD protein, approximately 9-12 Glu residues are converted to Gla. The propeptide and the Gla domain sequences are conserved and there is very little variation from species to species. Once carboxylation occurs, the propeptide is cleaved and the mature protein is then secreted from the cell. When the VKD protein is in the presence of calcium, it undergoes a conformational change allowing the Gla domain to bind to membrane surfaces, particularly those located near damaged vascular tissue. Once there, the Gla-containing proteins can promote and regulate blood coagulation.

The goal of my research involves analytically determining the active site for substrate binding to carboxylase. Using the understanding of carboxylase binding of Vitamin K dependent proteins, a 33-mer peptide was synthesized containing a tightly binding consensus propeptide region followed by a Gla domain sequence. Within this Gla domain, a photoactive crosslinker acts to strongly bind carboxylase adjacent to carboxylated Glu residues in the peptide. To further aid in the detection of binding, a fluorescent tag is added at the end of the peptide chain. After in vitro carboxylation of the synthesized peptide, the peptide-carboxylase subunit can be proteolytically digested and the fluorescent probe can be tracked and analyzed via ultra high pressure reversed-phase liquid chromatography and electrospray quadrupole-time of flight mass spectrometry.

Although there has been almost 30 years of extensive research performed on the behavior of gamma-carboxylase, there is still much more to be discovered to understand the mechanism of the enzyme, its relationship to cofactors, and its role in various protein substrates 3-4. Chromatographic methods have played a critical role in protein and peptide analyses because of its high sensitivity, minimal sample requirements, and ability to identify protein molecular weight and sequence when coupled to mass spectrometry. The use of capillary columns packed in-house with small particle diameters coupled with ultra-high pressure pumps to create gradient elution, aid in efficient separation and enhanced resolution between peptides of interest 5-7. Using chromatographic techniques, the biological problem of carboxylase’s active site can be further pursued providing additional information not only into the amino acid sequence for binding but also the behavior of this enzyme and its role in various biological functions.

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3. Mutucumarana, V.P.; Acher, F.; Straight, D.L.; Jin, D.; Stafford, D.W. 2003 J. Bio. Chem. 278(47): 46488-46493.
4. Tie, J.; Mutucumarana, V.P.; Straight, D.L.; Carrick, K.L.; Pope, R.M.; Stafford, D.W. 2003. J. Bio. Chem. 278(46): 45468-45475.
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7. Mellors, J.S.; Jorgenson, J.W. 2004 Anal. Chem. 76: 5441-5450.