Verbatim): The broad biological objective of this project is to continue studies to understand the complete vitamin K cycle, which is important for blood coagulation, bone formation, and a number of other physiological processes. The primary effort will continue to be studying the structure and function of gamma-glutamyl carboxylase and the mechanism of carboxylation. Another major element of the study will be to purify the microsomal enzyme vitamin K epoxide reductase, which is essential for regenerating vitamin K. The long-term goal of the research is to replicate not only the carboxylation process but the entire vitamin K cycle in vitro in order to understand the roles that carboxylase, vitamin K epoxide reductase, and any other essential components in the vitamin K cycle play in blood coagulation. Most of the research proposed in this application is devoted to understanding how the structure of the human gamma-glutamyl carboxylation enzyme is related to its function. Some overarching goals of the proposed research are: (1) determine the ways carboxylase recognizes and interacts with substrates and ligands: (2) identify and characterize catalytic residues crucial for gamma-glutamyl carboxylation; (3) elucidate the role of regulatory sites on the mechanism of gamma-glutamyl carboxylation; and (4) define a structural organization of the carboxylase enzyme by 2-dimensional electron diffraction. In particular, the Specific Aims are:
Specific Aim 1 : Identify sequences in the carboxylase that bind propeptide, investigate the role of carboxylase residues surrounding L394, which may constitute part of the gla domain-binding site, and determine which residues mediate linkage between these two sites.
Specific Aim 2 : Identify cysteine residues important for catalysis and functionally important disulfide bonds.
Specific Aim 3 : Investigate the relationship of the carboxylase's putative internal propeptide sequence to control of enzyme activity.
Specific Aim 4 : Examine what factors determine the substrate's rate of dissociation from the carboxylase in vitro and whether the rate of dissociation of substrate/product affects the extent of carboxylation of proteins expressed in cell culture.
Specific Aim 5 : Identify the gene for the vitamin K epoxide reductase.
Specific Aim 6 : Continue our collaboration with Dr. Kuhlbrandt's laboratory to obtain structural information about gamma-glutamyl carboxylase. The pursuit of these goals will include making chimeras of human and Drosophila carboxylases, using fluorescence spectroscopy to measure on- and off-rates of substrates used in carboxylation, and using the power of Drosophila genetics to attempt to identify the gene for epoxide reductase.
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