ENGINEERING MANGANESE METALLOENZYMES

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各自含有一个双核 L-精氨酸水解制L-鸟氨酸+尿素所需的锰(II)簇及其研究 表明催化作用是通过一种机制进行的，在该机制中，两种金属离子都能激活金属- 桥联氢氧离子作为催化亲核试剂。我们已经确定了人类的晶体结构 精氨酸酶I的分辨率为1.5A，这是迄今为止已确定的任何精氨酸酶的最高分辨率结构。 由于这种酶在多发性硬化症和癌症化疗中的作用，它是一种潜在的药物靶点 对于免疫反应，我们建议进行基于结构的抑制剂设计实验，这些实验可能会产生具有 亚纳摩尔亲和力，这将被用来探索精氨酸酶及其 与免疫反应中没有合成酶的关系。我们还提出了探索亲缘关系的实验 酶活性部位和结合底物之间的直接氢键和水介导氢键的重要性 或抑制剂。这些实验将使我们能够确定分子识别中的细微差别 在人类精氨酸酶I和II之间，可能在基于结构的同工酶设计中被利用- 特定的抑制剂。 此外，由于新发现的和意想不到的精氨酸酶和组蛋白之间的结构关系 脱乙酰酶，我们建议用来确定人组蛋白的位点特异性变体的X射线晶体结构 脱乙酰酶-8，并将这些结构与酶学测量相关联。因为这种酶是一种已被证实的 作为肿瘤化疗的靶点，对结构-功能关系的详细了解对于 推进可能产生新化疗药物的新抑制剂设计的探索。虽然组蛋白 脱乙酰酶采用与精氨酸酶相同的折叠，精氨酸酶的双核锰位只对应于一个 组蛋白脱乙酰酶中的单核锌位点，表明这两种金属酶的分化进化 来自原始的金属酶前体。我们提议的研究将突出机械论上的相似之处 在这两种金属水解酶之间，我们的研究也将指明组蛋白脱乙酰基酶 机制与细菌脱乙酰酶的机制有关，细菌脱乙酰酶需要二价锌或铁来 功能。

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.