ENGINEERING MANGANESE METALLOENZYMES

为了促进我们对锰金属酶大家族的理解，我们继续 着重于酶的结构-机理关系。精氨酸酶I和II各自含有一个双核 锰（II）簇所需的L-精氨酸水解形成L-鸟氨酸加尿素，我们的研究 表明催化作用是通过一种机制进行的，在这种机制中，两种金属离子都起到激活金属的作用， 桥连氢氧根离子作为催化亲核试剂。我们已经确定了人类的晶体结构 在1.5 A分辨率下，这是迄今为止确定的任何内切酶的最高分辨率结构。 由于这种酶是多发性硬化症和癌症化疗的潜在药物靶点， 免疫反应，我们提出了基于结构的走廊设计实验，可能会产生抑制剂， 亚纳摩尔的亲和力，这反过来将被用来探索的生物学功能， 与免疫反应中NO合酶的关系。我们还提出了实验，以探讨相对 酶活性位点和结合底物之间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 在人的同工酶I和II之间，可能在基于结构的同工酶设计中被利用- 特异性抑制剂 此外，鉴于新发现的和意想不到的结构关系之间的酶和组蛋白， 脱乙酰酶，我们建议确定X射线晶体结构的位点特异性变体的人类组蛋白 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 作为癌症肿瘤化疗的靶点，对结构-功能关系的详细了解对于 推进新的抑制剂设计的探索，可能会产生新的化疗药物。尽管组蛋白 脱乙酰酶采用与脱乙酰酶相同的折叠，脱乙酰酶的双核锰位点仅对应于一个 组蛋白脱乙酰酶中的单核锌位点，表明这两种金属酶的不同进化 从原始金属酶前体。我们提出的研究将突出机械的相似之处 在这两种金属水解酶之间，我们的研究也将表明组蛋白去乙酰化酶是如何 机制与细菌脱乙酰酶的机制相关，细菌脱乙酰酶需要二价锌或铁， 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.