STRUCTURE AND MECHANISM OF E COLI D-MANNITOL PERMEASE

大肠杆菌D-甘露醇渗透酶的结构与机制

• 批准号: 3275530
• 负责人: GARY R JACOBSON
• 金额: $ 12.18万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-07-01 至 1989-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3275530
• 关键词: Escherichia coli; active transport; allosteric site; autoradiography; carbohydrate transport; conformation; enzyme induction /repression; enzyme substrate; genetic manipulation; intercellular connection; mannitol; membrane permeability; microorganism metabolism; phospholipids; phosphorylation; plasmids; pyridoxal phosphate; pyruvate kinase; radiotracer; stereochemistry

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.

我们实验室正在积极研究其结构和机制 酶/渗透负责紧密耦合的运输和 D-甘露醇在大肠杆菌中的磷酸化。 我们的结果表明 该蛋白质：1）不对称地跨越大肠杆菌的内膜 其质量的很大一部分暴露于细胞的细胞质； 2) 可能在其底物运输过程中被共价磷酸化； 3）被钒酸盐抑制，可能模拟过渡态 酶催化的磷酸转移过程中的磷酸基团； 4）是 对 D-甘露醇具有高度但不是绝对的特异性； 5）具体是 在某些条件下被吡哆醛 5'-磷酸（一种化合物）抑制 它可能类似于酶的磷酸组氨酸底物；和 6) 是 由许多磷酸化合物以变构方式激活，包括 无机磷酸盐、磷酸烯醇丙酮酸、ADP 和 AMP。 在提议的 项目继续进行，我们的目标是：1）确定领域 暴露于膜外表面的多肽， 嵌入膜内，并暴露于膜内表面； 2） 确定这些域在构象过程中是否以及如何改变 催化循环； 3) 测定甘露醇的立体化学过程 酶的磷酸化； 4) 肽的分离和表征 在蛋白质的不同位点进行专门标记； 5) 确定 的变构调节的性质和生理意义（如果有的话） 酶。 生物化学和膜生理学的结合 这些调查中将使用一些方法。 顺利完成 这些研究将在实现我们的总体目标方面取得重大进展 了解该系统中运输的分子基础及其 规定。 因为所有细胞都依赖于高效的主动运输系统 为了他们的生计和细胞间通讯过程， 分子水平的运输机制的知识对于 了解正常和正常情况下的生长和新陈代谢原理 患病细胞。