STRUCTURE AND MECHANISM OF E COLI D-MANNITOL PERMEASE

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Our laboratory is actively investigating the structure and mechanism of the enzyme/permease responsible for the tightly coupled transport and phosphorylation of D-mannitol in Escherichia coli. Our results have shown that this protein: 1) spans the inner membrane of E. coli asymmetrically with a large proportion of its mass exposed to the cytoplasm of the cell; 2) is probably covalently phosphorylated during transport of its substrate; 3) is inhibited by vanadate which may mimic a transition state of the phosphate group during phosphotransfer catalyzed by the enzyme; 4) is highly, but not absolutely, specific for D-mannitol; 5) is specifically inhibited under certain conditions by pyridoxal 5'-phosphate, a compound which may resemble a phospho-histidine substrate of the enzyme; and 6) is activated allosterically by a number of phospho-compounds including inorganic phosphate, phosphoenolpyruvate, ADP and AMP. In the proposed project continuation, our aims are to: 1) determine the domains of the polypeptide that are exposed to the exterior surface of the membrane, embedded in the membrane, and exposed at the interior membrane surface; 2) determine if and how these domains change in conformation during the catalytic cycle; 3) determine the stereochemical course of mannitol phosphorylation by the enzyme; 4) isolate and characterize peptides specifically labeled at various sites on the protein; and 5) determine the nature and physiological significance, if any, of allosteric regulation of the enzyme. A combination of biochemical and membrane physiological approaches will be used in these investigations. Successful completion of these studies will lead to significant progress toward our overall goal of understanding the molecular basis of transport in this system and its regulation. Because all cells rely on efficient active transport systems for their livelihood and for intercellular communication processes, a knowledge of transport mechanism at the molecular level is essential in understanding the principles of growth and metabolism in both normal and diseased cells.