ENGINEERING MANGANESE METALLOENZYMES

工程锰金属酶

• 批准号: 7168416
• 负责人: DAVID W CHRISTIANSON
• 金额: $ 25.58万
• 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/7168416
• 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

DESCRIPTION (provided by applicant): 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-arginine to 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 inhibitor design 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 divalent zinc or iron for function.

描述（由申请人提供）：为了促进我们对更大的锰金属酶家族的理解，我们继续专注于精氨酸酶的结构机理关系。精氨酸酶I和II都包含L-精氨酸水解以形成L- or氨酸加尿素所需的双核锰（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，两种金属离子都可以激活金属纤维纤维的氢氧化物，将其作为催化核定型。我们已经确定了1.5 A分辨率的人精氨酸酶I的晶体结构，这是迄今为止确定的任何精氨酸酶的最高分辨率结构。由于该酶是由于其在免疫反应中的作用而导致多发性硬化症和癌症化疗的潜在药物靶标，因此我们提出了基于结构的抑制剂设计实验，可能会产生具有亚纳米尔亲和力的抑制剂，进而将其用于探索精氨酸酶的生物学功能及其在免疫反应中没有合成酶的关系。我们还提出了实验，以探测酶活性位点和结合底物或抑制剂之间直接和水介导的氢键的相对重要性。这些实验将使我们能够确定人精氨酸酶I和II之间的分子识别差异，这些差异可能在同工酶特异性抑制剂的基于结构的设计中可能被利用。此外，鉴于精氨酸酶和组蛋白脱乙酰基酶之间新发现的和意外的结构关系，我们建议确定人组蛋白脱乙酰基酶8的位点特异性变体的X射线晶体结构，并将这些结构与酶学测量相关联。由于该酶是癌症肿瘤化疗的验证靶标，因此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至关重要。尽管组蛋白脱乙酰基酶采用与精氨酸酶相同的折叠，但精氨酸酶的双核锰位点仅对应于组蛋白脱乙酰基酶中的单核锌位点，这表明这两种金属酶从原始的金属酶前体的发散演化。我们提出的研究将突出这两种金属水合酶之间的机械相似之处，我们的研究还将指出组蛋白脱乙酰基酶的机理与需要二价锌或铁的功能的细菌脱乙酰基酶的机制如何相关。