Vitamin K is required for blood clotting where it is an essential cofactor in the complex enzymic sequence known as the blood clotting cascade. Together with a vitamin K-dependent carboxylase, carbon dioxide and molecular oxygen, vitamin K effects the carboxylation of selected glutamate residues in a number of proteins in the blood clotting sequence. These include: factor II (prothrombin), factor VII, factor IX, factor X and protein C, S and Z; as well as the bone protein osteocalcin. The carboxylation reaction is essential to enable calcium binding to membrane surfaces of zymogen precursors to the blood cascade enzymes. In the absence of carboxylation blood clotting is inhibited. The significance of vitamin K lies in its mechanism of action and its interaction with clinically important anticoagulants. The research proposed in this application will explore enzyme experiments based on a new experimental model for the vitamin K-dependent carboxylation. On the basis of recent model experiments, it is suggested that molecular oxygen triggers the carboxylation event by reacting with the anionic form of vitamin K hydroquinone to produce vitamin K oxide. Formation of the latter provides the energy necessary to furnish a base sufficiently strong to effect the carboxylation. Experiments with 18-O2 will probe the involvement of a special dioxetane intermediate. This experiment will then be extended to define the active site of vitamin K. The mechanism of action of active analogues of the vitamin K-dependent carboxylase will be explored and new potential inhibitors will be synthesized. A new mechanism of action of vitamin K3 in controlling tumor cell growth will be explored.
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