REACTION MECHANISMS FOR ENZYMES

酶的反应机制

• 批准号: 3486036
• 负责人: JULES Alan SHAFER
• 金额: $ 23.04万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3486036
• 关键词: Escherichia coli; calcium; chemical fingerprinting; chemical structure function; cysteine; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate complex; fibrin; fibrin stabilizing factor; fibrinogen; glycine; high performance liquid chromatography; human subject; human tissue; molecular pathology; nuclear magnetic resonance spectroscopy; protein engineering; protein sequence; site directed mutagenesis; thrombin; thrombosis

Papain, a thiol protease from papaya latex, will be studied to determine the role of Asp-158 in catalysis and to determine the effect of Asp-158 on the interactive ionization of His-159 and Cys-25 at the active site of papain. Derivatives of papain modified at Asp-158 will be prepared and characterized with respect to their altered interactions with substrates and inhibitors. Proton NMR and potentiometric difference titrations will be used to determine alterations in the ionization behavior of His-159 and Cys-25 caused by modification of Asp-158. The interactive ionization of His-159 and Cys-25 also will be investigated to determine the effect of the ion-pair interaction on the reactivity of these residues. The nucleophilic reactivity of ammonium-thiolate ion-pairs in nonenzymic reactions will be studied to evaluate the possible catalytic advantage of the imidazolium-thiolate ion-pair at the active site of papain. The potentiometric difference titration method we have developed for determining the ionization behavior of the thiol group in papain will be used to determine the possible existence of ligand dependent ionic interactions involving Cys-beta 93 of hemoglobin. D-Serine dehydratase from E. coli will be studied to determine a) how monovalent cations effect the affinity of the enzyme for its cofactor pyridoxal 5'-phosphate, b) the involvement of a thiol group in the catalytic activity of the enzyme, and c) the intermediates in the catalytic pathway and their rates of interconversion. Studies with human fibrinogen from individuals with dysfibrinogenemia are proposed in which amino acid replacements in abnormal fibrinogens will be related to their altered functional competence especially their altered interactions with the enzymes involved in blood clot formation dissolution.

木瓜蛋白酶，一种来自木瓜胶乳的巯基蛋白酶，将被研究以确定 Asp-158在催化中的作用，并确定Asp-158对 His-159和Cys-25在木瓜蛋白酶活性位点的相互作用电离。 将制备和表征在Asp-158处修饰的木瓜蛋白酶衍生物 关于它们与底物和抑制剂的改变的相互作用。 将使用质子NMR和电位差滴定法测定 His-159和Cys-25电离行为的改变， Asp-158的修饰。 His-159和Cys-25的相互作用电离也 将进行研究，以确定离子对相互作用对 这些残余物的反应性。 硫醇铵的亲核反应性 离子对在非酶反应将进行研究，以评估可能的 咪唑-硫醇盐离子对在活性位点的催化优势 木瓜蛋白酶 本文介绍了我们研制的电位差滴定法 确定木瓜蛋白酶中巯基的电离行为将用于 确定可能存在的配体依赖性离子相互作用 包括血红蛋白的半胱氨酸β 93。 D-丝氨酸脱氢酶来源于E.大肠杆菌将进行研究，以确定a）如何单价 阳离子影响酶对其辅因子吡哆醛的亲和力 5 ′-磷酸酯，B）硫醇基参与的催化活性 所述酶，和c）催化途径中的中间体和它们的降解速率， 相互转换 对来自纤维蛋白原异常血症个体的人纤维蛋白原的研究如下： 提出了异常纤维蛋白原中的氨基酸替换将被 与他们的功能能力改变有关，特别是他们的 与参与血凝块形成溶解的酶相互作用。