The purpose of this project is to understand how the primary structure of vitamin K-dependent substrates controls their interaction with the gamma glutamyl carboxylase and to identify the gene of the related vitamin K epoxide reductase. These enzymes are responsible for the vitamin K dependent post-translational modification of proteins involved in hemostasis, bone metabolism and cell growth. Understanding the structure and function of these enzymes will certainly lead to clinical applications in the treatment of many areas of human disease.
The specific aims are: One. Initiate an entirely new approach to identify the vitamin K epoxide reductase. The genes for warfarin resistance and epoxide reductase apparently map to the same locus making it possible to localize the gene to a narrow region of human chromosome 16. Thus one can use a direct assay to ?knock out? specific messenger RNA molecules with small double stranded RNA to identify the gene in cell culture. Two. Determine the residues of factor IX?s propeptide that bind to specific residues at the propeptide binding site of the carboxylase. A combination of traditional protein chemistry and phage display methods will be used. Three. Use capillary electrophoresis coupled with mass-spectrometry to determine the order of the addition of carbon dioxide to the twelve Gla residues of the Gla domain of factor IX. The hypothesis is that the glutamic acids most critical for functions will be carboxylated first. Four. Continue to examine the mechanism of carboxylation using various substrates. The role of the Gla domain will be examined by utilizing substrates that have the same propeptide but different Gla domains. These experiments will be done in enzyme excess using pre steady state conditions. The interaction between the propeptide and Gla domain will be examined by utilizing substrates that have the same Gla domain but different propeptides.
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