ENGINEERING MANGANESE METALLOENZYMES

描述(申请人提供)：为了促进我们对锰金属酶大家族的了解，我们继续关注精氨酸酶的结构-机制关系。精氨酸酶I和精氨酸酶II都含有一个双核锰(II)簇，这是L-精氨酸水解生成L-鸟氨酸+尿素所必需的，我们的研究表明，催化作用是通过一种机制进行的，在该机制中，两种金属离子都激活了金属桥联的氢氧离子作为催化亲核试剂。我们在1.5A分辨率下测定了人精氨酸酶I的晶体结构，这是迄今为止所确定的精氨酸酶的最高分辨率结构。由于这种酶在免疫反应中的作用，它是治疗多发性硬化症和癌症化疗的潜在药物靶点，我们建议进行基于结构的抑制剂设计实验，以产生亚纳摩尔亲和力的抑制剂，进而用于探索精氨酸酶的生物学功能及其与免疫反应中一氧化氮合酶的关系。我们还建议进行实验，以探索酶活性部位和结合底物或抑制剂之间的直接氢键和水介导氢键的相对重要性。这些实验将使我们能够确定人类精氨酸酶I和II之间分子识别的细微差异，这些差异可能被用于基于结构的同工酶特异性抑制剂的设计。此外，考虑到精氨酸酶和组蛋白脱乙酰酶之间新发现的和意想不到的结构关系，我们建议确定人组蛋白脱乙酰酶-8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。由于该酶已被证实是癌症肿瘤化疗的靶点，因此对结构-功能关系的详细了解对于推进可能产生新的化疗药物的新的抑制剂设计的探索至关重要。尽管组蛋白脱乙酰酶与精氨酸酶采用相同的折叠，但精氨酸酶的双核锰位仅对应于组蛋白脱乙酰酶中的单核锌位，这表明这两种金属酶从原始金属酶前体分化进化而来。我们拟议的研究将突出这两种金属水解酶之间的机制相似之处，我们的研究还将指明组蛋白脱乙酰酶机制如何与细菌脱乙酰酶机制相关，细菌脱乙酰酶机制需要二价锌或铁才能发挥作用。

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.