ENGINEERING MANGANESE METALLOENZYMES

工程锰金属酶

• 批准号: 7347586
• 负责人: DAVID W CHRISTIANSON
• 金额: $ 25.59万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7347586
• 关键词: Acids; Active Sites; Adopted; Affinity; Anabolism; Arginine; Binding Sites; Biological; Biological Process; Biology; Boronic Acids; Catalysis; Chemicals; Chemistry; Chemotherapy-Oncologic Procedure; Class; Clinical; Complex; Deacetylase; Drug Delivery Systems; Engineering; Enzymatic Biochemistry; Enzymes; Evolution; Family; Fluorides; Funding; Goals; Histone Deacetylase; Human; Hydrogen Bonding; Hydrolysis; Hydroxide Ion; Immune response; Ions; Iron; Isoenzymes; L Forms; Malignant Neoplasms; Manganese; Measurement; Mediating; Medical; Medicine; Metals; Molecular; Mononuclear; Multienzyme Complexes; Multiple Sclerosis; Muscle relaxation phase; Nature; Neutrons; Nitric Oxide Synthase; Ornithine; Physicians; Physiology; Play; Process; Rattus; Recombinants; Relative (related person); Research; Resolution; Roentgen Rays; Role; Scientist; Sexual Dysfunction; Site; Specificity; Structure; Structure-Activity Relationship; Transition Elements; Urea; Variant; Water; Woman; Work; Zinc; arginase; base; biomedical scientist; boronic acid; carboxylate; chemotherapy; day; design; inhibitor/antagonist; men; metalloenzyme; molecular recognition; novel; programs; research study; stoichiometry; structural biology; success; therapeutic target; thiosemicarbazide; tumor

In order to advance our understanding of the greater family of manganese metalloenzymes, we continue to focus on structure-mechanism relationships in arginase. Arginases I and II each contain a binuclear manganese(II) cluster required for the hydrolysis of L-arginineto form L-ornithine plus urea, and our studies indicate that catalysis proceeds through a mechanism in which both metal ions function to activate a metal- bridging hydroxide ion as the catalytic nucleophile. We have determined the crystal structure of human arginase I at 1.5 A resolution, and this is the highest resolution structure of any arginase determined to date. Since this enzyme is a potential drug target for multiple sclerosis and cancer chemotherapy due to its role in the immune response, we propose structure-based inhibitordesign experiments that may yield inhibitors with sub-nanomolar affinity, which in turn will be used to explore the biological function of arginase and its relationships with NO synthase in the immune response. We, also propose experiments to probe the relative importance of direct and water-mediated hydrogen bonds between the enzyme active site and bound substrate or inhibitors. These experiments will allow us to determine subtle differences in molecular recognition between human arginases I and II that may potentially be exploited in the structure-based design of isozyme- specific inhibitors. Additionally, given the newly-discovered and unexpected structural relationship between arginase and histone deacetylase, we propose to determine X-ray crystal structures of site-specific variants of human histone deacetylase-8 and correlate these structures with enzymological measurements. Since this enzyme is a proven target for cancer tumor chemotherapy, a detailed understanding of structure-function relationships is critical to advance the exploration of new inhibitor designs that may yield novel chemotherapeutics. Although histone deacetylase adopts an identical fold to arginase, the binuclear manganese site of arginase corresponds to only a mononuclear zinc site in histone deacetylase, indicative of divergent evolution of these two metalloenzymes from a primordial metalloenzyme precursor. Our proposed studies will highlight the mechanistic parallels between these two metallohydrolases, and our studies will also indicate how the histone deacetylase mechanism correlates with the mechanisms of bacterial deacetylases that require divalentzinc or iron for function.

为了促进我们对大型锰金属酶家庭的理解，我们继续 专注于精氨酸酶中的结构机制关系。精氨酸酶I和II都包含一个双核 L-Arginineto的水解L- or-氨酸氨酸加尿素所需的锰（II）簇，我们的研究 表明催化是通过一种机制进行的，在该机制中，两种金属离子都在激活金属 将氢氧化离子桥接为催化亲核试剂。我们已经确定了人类的晶体结构 精氨酸酶I的分辨率为1.5，这是确定迄今确定的任何精氨酸酶的最高分辨率结构。 由于该酶是多发性硬化症和癌症化疗的潜在药物，因为它在 免疫反应，我们提出了基于结构的抑制剂实验，该实验可能会产生抑制剂 亚纳摩尔亲和力，这将用于探索精氨酸酶及其其生物学功能 免疫反应中无合酶的关系。我们还提出了实验以探测相对 酶活性位点和结合底物之间的直接和水介导的氢键的重要性 或抑制剂。这些实验将使我们能够确定分子识别的细微差异 在人类精氨酸酶I和II之间，在同工酶的基于结构的设计中可能被利用 特定抑制剂。 此外，考虑到精氨酸酶与组蛋白之间的新发现和意外的结构关系 脱乙酰基酶，我们建议确定人组蛋白的位点特异性变体的X射线晶体结构 脱乙酰基酶8并将这些结构与酶学测量相关。由于这种酶是一种经过验证的 癌症肿瘤化疗的靶点，对结构功能关系的详细理解对于 推进可能产生新型化学治疗剂的新抑制剂设计的探索。虽然组蛋白 脱乙酰基酶采用与精氨酸酶相同的折叠，精氨酸酶的双核锰部位仅对应于 组蛋白脱乙酰基酶中的单核锌位点，指示这两个金属酶的发散进化 来自原始金属酶前体。我们提出的研究将突出机械的相似之处 在这两种金属水合酶之间，我们的研究还将指示组蛋白脱乙酰基酶如何 机制与需要脱甲替津或铁的细菌脱乙酰基酶的机制相关 功能。