STRUCTURE AND MECHANISM OF E COLI D-MANNITOL PERMEASE

我们实验室正在积极研究该蛋白的结构和机制。 酶/渗透酶负责紧密耦合的运输和 D-甘露醇在大肠杆菌中的磷酸化。我们的结果显示 这种蛋白质：1)不对称地跨过大肠杆菌的内膜 其很大一部分质量暴露在细胞质中； 2)在底物运输过程中可能发生共价磷酸化； 3)被可能模拟过渡状态的钒酸盐抑制 酶催化的磷酸转移过程中的磷酸基团；4) 对D-甘露醇高度专一，但不是绝对专一；5)专一 在一定条件下被5‘-磷酸吡哆醛抑制，一种化合物 其可类似于酶的磷酸组氨酸底物；以及6) 被一些含磷化合物变构激活，包括 无机磷、磷酸烯醇式丙酮酸、ADP和AMP。在建议的 项目继续，我们的目标是：1)确定 暴露在膜外表面的多肽， 嵌入膜内，并暴露在膜内表面；2) 确定这些结构域是否以及如何在构象上发生变化 催化循环；3)测定甘露醇的立体化学过程 酶的磷酸化；4)多肽的分离和鉴定 在蛋白质上的不同位置特别标记；以及5)确定 变构调节的性质和生理意义，如果有的话 这种酶。生化与膜生理的结合 在这些调查中将使用各种方法。成功完成 这些研究将导致我们朝着以下总目标取得重大进展 了解这一系统中运输的分子基础及其 监管。因为所有细胞都依赖于高效的主动运输系统 为了它们的生计和细胞间的通讯过程， 在分子水平上的转运机制的知识是必不可少的 了解正常儿童和正常儿童的生长和代谢规律 病态细胞。

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.