Prostanoids are oxygenated metabolites of polyunsaturated fatty acids which have major roles in vascular pathophysiological processes, including hemostasis, thrombosis, and stroke. Synthesis of the prostanoids involves the sequential actions of several enzymes, most of them membrane associated. One of these enzymes, thromboxane A synthase (TXAS), catalyzes the isomerization of prostaglandin H2 into thromboxane A2, a potent vasoconstrictor and inducer of platelet aggregation. TXAS also catalyzes the fragmentation of prostaglandin H2 to malodialdehyde and 12-hydroxyheptatrienoic acid. TXAS is found in many human tissues, primarily in monocytoid and tissue macrophages. Although TXAS is recognized to be membrane associated, its exact intracellular location and the arrangement of its polypeptide chain with respect to the membrane remain to be established. TXAS is a member of the cytochrome P450 superfamily, but it lacks the monooxygenase activity typical of cytochrome P450s. TXAS has a conserver cysteine residue which is likely to be the proximal ligand for the heme prosthetic group, but very little is known about other residues making up the TXAS active site and their roles in the two reactions catalyzed. The overall goal of this project is to understand the catralytic and regulatory functioning of human TXAS in terms of its structure, and of the interactions of the protein with its native membrane environment.
The specific aims are: 1) Determine the subcellular localizatioin and membrane topology of TXAS; and 2) Identify amino acid residues in TXAS important to heme and substrate binding, and to catalysis. Thetopological studies will determine which parts of the TXAS polypeptide are oriented towards the cytoplasm and which toward the lumenal compartment in the cell, and which parts serve as membrane anchor. The active site studies will elucidate the TXAS catalytic machinery. Both levels of structural information are needed to improve our grasp of thromboxane biosynthesis, and how it fits into the overall prostanoid biosynthesis process. Methodologies to be used include: immunofluorescence and immunoelectron microscopy; selective membrane permeabilization; mapping with site- specific antibodies; molecular modelling; proteolytic modification; analysis of enzymatic activity; site-directed mutagenesis; heterologous expression of mutant protein; and enzyme immunoassay.
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