STRUCTURE AND MECHANISM OF E COLI D-MANNITOL PERMEASE

我们的实验室正在积极研究的结构和机制， 负责紧密偶联转运的酶/通透酶， 大肠杆菌中D-甘露醇的磷酸化。 我们的结果显示 这种蛋白质：1）跨越E.大肠杆菌不对称 其大部分质量暴露于细胞的细胞质; 2)可能在其底物转运过程中共价磷酸化; 3)被钒酸盐抑制，钒酸盐可以模拟 在酶催化的磷酸转移过程中， 对D-甘露醇具有高度特异性，但不是绝对特异性; 5）特异性 在某些条件下被吡哆醛5 '-磷酸盐抑制， 其可能类似于酶的磷酸-组氨酸底物;和6） 被许多磷化合物变构激活，包括 无机磷酸盐、磷酸烯醇丙酮酸盐、ADP和AMP。 拟议 项目的延续，我们的目标是：1）确定域的 暴露于膜外表面的多肽， 包埋在膜中，并暴露在膜的内表面; 2） 确定这些结构域是否以及如何在构象中变化， 催化循环; 3）确定甘露醇的立体化学过程 通过酶磷酸化; 4）分离和表征肽 在蛋白质上的各个位点特异性标记;和5）确定 性质和生理意义，如果有的话，变构调节 酶 生物化学和膜生理学的结合 在这些调查中将使用各种方法。 成功完成 这些研究将使我们的总体目标取得重大进展， 了解该系统中运输的分子基础及其 调控 因为所有的细胞都依赖于有效的主动运输系统 为了他们的生计和细胞间的通讯过程， 在分子水平上的运输机制的知识是必不可少的， 了解生长和代谢的原则，在正常和 病变细胞

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.