MECHANISM OF INOSITIDE-RELATED ENZYMES

肌醇相关酶的作用机制

• 批准号: 2175756
• 负责人: MING-DAW TSAI
• 金额: $ 20.33万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-07-01 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2175756
• 关键词: Bacillus; Escherichia coli; annexins; bacterial proteins; enzyme complex; enzyme mechanism; enzyme structure; enzyme substrate analog; gene expression; high performance liquid chromatography; inositol phosphates; nuclear magnetic resonance spectroscopy; oxygen; phosphatidylinositols; phospholipase C; phospholipase inhibitor; radiotracer; site directed mutagenesis; stable isotope; stereoisomer; structural biology

The inositol-related pathways are extremely complicated and important biologically. The key step in these pathways is the conversion of phosphatidylinositol (PI) to diacylglycerol, inositol 1,2-cyclic phosphate (IcP), and inositol 1-phosphate (IP), catalyzed by PI-specific phospholipase C (PI-PLC). The goal for the next period is to extend our stereochemical studies of PI-PLC to structure-function analyses of three enzymes which catalyze three variations of this process: bacterial PI-PLC from B. thuringiensis, mammalian PI-PLC-beta1 from bovine brain, and annexion III from human lung. Annexion III is a Ca2+-dependent phospholipid binding protein in the lipocortin/annexion family but has been demonstrated to catalyze reactions similarly to PI-PLC except that the substrate is glycerophosphoinositol (GroPI) instead of Pl. The long range goals are to understand how these enzymes catalyze their reactions, and what aims for the next granting period: (1) To overexpress annexion II in E. coli. The B. thuringiensis PI-PLC will be purified from an overproducing E. coli strain given to us by Dr. Henner at Genentech. (2) To develop continuous assay methods, and to establish the basic kinetic properties of the three enzymes. (3) To synthesize three types of substrate analogs, with modifications on the inositol moiety: deoxy analogs, configurational isomers, and epoxy analogs. (4) To probe the substrate specificity of all three enzymes using the deoxy analogs of substrates. (5) To probe the stereospecificity of all three enzymes using the configuration a isomers of substrates. The steric courses of the reactions catalyzed by annexion III will also be elucidated by employing 170 and 180 isotopes. (6) To test the epoxy analogs of PI and GroPI as irreversible inhibitors. The potent inhibitors will then be synthesized in radioactive forms and used to label the enzyme. The labelled enzymes will be digested and the site of modification identified. (7) To initiate site-directed mutagenesis studies with the bacterial Pi-PLC and annexion III. The first target residues will be chosen based on sequence homology, the results of chemical modification, and crystal structures from other laboratories. (8) To attempt proton NMR analyses, and to try growing crystals, of the free enzymes and enzyme-inhibitor complexes, for bacterial PI-PLC and annexion III (both with molecular weight ca. 35,000).

与肌醇相关的途径非常复杂且重要 生物学上。 这些途径的关键步骤是转换 磷脂酰肌醇（PI）至二酰基甘油，肌醇1,2-偶然醇 磷酸盐（ICP）和肌醇1-磷酸（IP），由PI特异性催化 磷脂酶C（PI-PLC）。 下一个时期的目标是扩展我们 PI-PLC对三个结构功能分析的立体化学研究 催化此过程的三个变化的酶：细菌PI-PLC 来自苏云金芽孢杆菌，哺乳动物pi-plc-beta1来自牛脑， 来自人类肺的附件III。 附件III是Ca2+依赖性的 脂皮质素/附件家族中的磷脂结合蛋白，但具有 被证明可以催化反应类似于PI-PLC，除了 底物是甘油磷酸肌醇（Gropi）而不是PL。 长 范围目标是了解这些酶如何催化其反应， 以及旨在下一个授予期的目的：（1）过表达附件 II在大肠杆菌中。 苏云金芽孢杆菌pi-plc将从 Genentech Henner博士向我们提供了过量生产大肠杆菌菌株。 （2） 开发连续的测定方法，并建立基本动力学 三种酶的特性。 （3）合成三种类型的 底物类似物，对肌醇部分进行了修改：脱氧 类似物，构型异构体和环氧类似物。 （4）探测 使用所有三种酶的底物特异性，使用的脱氧类似物 基材。 （5）使用使用所有三种酶的立体特异性 基板的配置A异构体。 的空间课程 附件III催化的反应也将通过采用 170和180同位素。 （6）测试Pi和Gropi的环氧类似物作为 不可逆的抑制剂。 然后将合成有效的抑制剂 以放射性形式，用于标记酶。 标记的酶 将被消化，并确定修改部位。 （7）至 通过细菌PI-PLC和 附件三。 将根据序列选择第一个目标残基 同源性，化学修饰的结果和晶体结构 来自其他实验室。 （8）尝试质子NMR分析并尝试 生长的晶体，自由酶和酶抑制剂复合物的生长晶体，用于 细菌PI-PLC和附件III（均具有分子量Ca。 35,000）。