REACTION MECHANISMS FOR ENZYMES

酶的反应机制

• 批准号: 3343199
• 负责人: JULES Alan SHAFER
• 金额: $ 18.12万
• 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 1989-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3343199
• 关键词: Bohr effect; Escherichia coli; afibrinogenemia; ammonium compounds; aspartate; chemical fingerprinting; chemical structure function; cofactor; cysteine; enzyme model; enzyme structure; enzyme substrate complex; fibrinogen; hemoglobin; histidine; human tissue; imidazole; ionization; ligands; molecular pathology; nuclear magnetic resonance spectroscopy; papain; plants; pyridoxal phosphate; stop flow technique; thiols

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.

木瓜蛋白酶是一种从木瓜乳汁中提取的硫醇蛋白酶，将用于测定木瓜乳胶中的 Asp-158在催化反应中的作用及Asp-158对 His-159和Cys-25在木瓜蛋白酶活性中心的相互作用电离。 在Asp-158上修饰的木瓜酶的衍生物将被制备和表征 关于它们与底物和抑制剂相互作用的改变。 将使用质子核磁共振和电位差滴定来确定 His-159和Cys-25的电离行为的改变 Asp-158的改性。His-159和Cys-25的相互作用电离 将被调查以确定离子对相互作用对 这些残留物的反应性。硫代铵的亲核反应性 非酶反应中的离子对将被研究以评估可能的 咪唑-硫代离子对在活性中心的催化优势 木瓜酶。 我们开发的电位差滴定法用于测定 测定木瓜蛋白酶中硫醇基团的电离行为将被用于 确定可能存在的依赖于配体的离子相互作用 涉及半胱氨酸-β93的血红蛋白。 来自大肠杆菌的D-丝氨酸脱水酶将被研究以确定a)如何单价 阳离子影响酶对辅因子吡哆醛的亲和力 5‘-磷酸，b)硫醇基团参与的催化活性 酶，以及c)催化途径中的中间体及其速率 相互转换。 对纤维蛋白原血症患者的人类纤维蛋白原的研究 建议将异常纤维蛋白原中的氨基酸替换 与他们改变的功能能力有关，特别是与他们改变的 与参与血液凝块形成和溶解的酶的相互作用。