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.

我们的实验室正在积极调查 负责紧密耦合的运输和 大肠杆菌中D-甘露醇的磷酸化。 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 其大量质量暴露于细胞的细胞质； 2）在其底物运输过程中可能会共价磷酸化； 3）被钒酸盐抑制，这可能模仿 在磷酸转移过程中，磷酸盐组由酶催化； 4）是 高度但不是绝对的，特有的是甘露醇； 5）是专门的 在某些条件下被吡ido醇5'-磷酸抑制，一种化合物 可能类似于酶的磷酸硫醇基质； 6）是 通过许多磷酸化合物（包括） 无机磷酸盐，磷酸烯醇丙酮酸，ADP和AMP。 在提议中 项目延续，我们的目标是：1）确定 暴露于膜外表面的多肽， 嵌入膜中，暴露在内膜表面； 2） 确定这些领域在构象中的变化以及如何变化 催化循环； 3）确定甘露醇的立体化学课程 酶磷酸化； 4）分离和表征肽 在蛋白质上的各个位点特别标记； 5）确定 自然和生理意义（如果有的话） 酶。 生化和膜生理的结合 这些调查将使用方法。 成功完成 这些研究将导致我们的整体目标取得重大进展 了解该系统及其运输的分子基础 规定。 因为所有电池都依赖有效的主动运输系统 对于他们的生计和细胞间交流过程， 在分子水平上了解运输机制的知识至关重要 了解正常和 病细胞。