The long-term objective of this research is to understand the complete vitamin K cycle in terms of structure and function. This series of reactions is important for post- translational modification of proteins involved in blood coagulation, bone formation, and a number of other physiological processes. The primary focus will be studying the structure and function of the enzyme, 3-glutamyl carboxylase, and its mechanism of action. The broader goal is to reproduce the carboxylation process, but in addition, the entire vitamin K cycle in vitro. This should help to understand the roles that carboxylase, vitamin K oxidoreductase, and any other essential components in the vitamin K cycle play in physiologic settings. Primarily, these studies will be designed to address the following: (1) determine the ways carboxylase recognizes and interacts with substrates and ligands; (2) identify and characterize catalytic residues crucial for 3-carboxylation; (3) elucidate the role of regulatory sites on the mechanism of 3-carboxylation; and (4) determine the three-dimensional structure of the 3-glutamyl carboxylase. In particular, the specific aims are as follows:
Specific Aim 1 : Investigate the role of substrate affinity in the control of vitamin K-dependent protein carboxylation in mammalian cells.
Specific Aim 2 : Identify residues important for substrate binding, propeptide binding, and the residues that mediate the allosteric interaction between these two sites; develop models compatible with our results to explain how carboxylase functions.
Specific Aim 3 : Identify the active site catalytic residues of carboxylase.
Specific Aim 4 : Investigate the three-dimensional structure of 3-glutamyl carboxylase. The pursuit of these goals will include creation of mutant carboxylases and biochemical characterization of these modified enzymes; use of cell culture and molecular biology techniques to investigate expression of pro-coagulation factors, with different affinities for carboxylase to study how carboxylation is controlled in a cellular milieu; and use electron crystallography to gain structural information that we will correlate with information from our functional studies to achieve a more complete understanding of the 3-glutamyl carboxylase. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL048318-15A2
Application #
7380124
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Link, Rebecca P
Project Start
1992-04-01
Project End
2011-11-30
Budget Start
2007-12-26
Budget End
2008-11-30
Support Year
15
Fiscal Year
2008
Total Cost
$362,162
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
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Gui, Tong; Reheman, Adili; Funkhouser, William K et al. (2007) In vivo response to vascular injury in the absence of factor IX: examination in factor IX knockout mice. Thromb Res 121:225-34
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