Enzymology of Dimethylargininase

描述（由申请人提供）：一氧化氮（NO）是一种在癌症生物学中起重要作用的小活性自由基。NO和其他氧化剂的异常产生存在于各种类型的肿瘤中，包括乳腺癌，也存在于慢性炎症部位，这与人类癌症风险的增加有关。此外，一氧化氮和其他氧化剂在缺血性中风期间引起神经元损伤。人类可以通过使用内源性NO合成酶抑制剂，单甲基和二甲基精氨酸来调节NO的产生。这些抑制剂的浓度依次由二甲基精氨酸酶的两种组织特异性异构体DDAH-1和DDAH-2控制，它们水解这些抑制剂以消除对NO产生的抑制。这两种DDAH亚型代表了NO药理调控的有吸引力的靶点，但对于这些酶如何工作，它们如何响应氧化和亚硝化应激，以及它们是否可以被小分子选择性地抑制，我们知之甚少。本申请有三个具体目的：1)确定DDAH的催化机制；2)确定DDAH是否能受到与生物相关的活性氧或活性氮的调控；3)开发DDAH的抑制剂。这些研究将在纯化蛋白上完成，着眼于确定影响其生理作用的同种异构体之间的功能差异，并可用于设计选择性抑制剂。通过了解DDAH催化和调控背后的化学作用，我们将开发出具有治疗潜力的新生化工具，并将更多地了解DDAH在癌症生物学中的作用以及对氧化和亚硝化应激的一般反应。

DESCRIPTION (provided by applicant): Nitric oxide (NO) is a small reactive radical that plays a significant role in cancer biology. Aberrant production of NO and other oxidants is found in various types of tumors, including breast cancer, and also at sites of chronic inflammation, which have been associated with increased risk of human cancers. In addition, NO and other oxidants have been implicated in causing neuronal damage during ischemic stroke. Humans can regulate NO production by using endogenous inhibitors of NO synthase, monomethyl- and dimethylarginine. The concentrations of these inhibitors are controlled, in turn, by two tissue specific isoforms of dimethylargininase, DDAH-1 and DDAH-2, which hydrolyze these inhibitors to remove inhibition of NO production. These two DDAH isoforms represent attractive targets for pharmacological manipulation of NO, but not much is known about how these enzymes work, how they respond to oxidative and nitrosative stress, and whether they can be inhibited selectively by small molecules. This application has three specific aims 1) to determine the catalytic mechanism of DDAH, 2) to determine whether DDAH can be regulated by biologically relevant reactive oxygen or nitrogen species, and 3) to develop inhibitors of DDAH. These studies will be completed on purified proteins with an eye toward determining functional differences between isoforms that would impact their physiological roles and that can be exploited for design of selective inhibitors. By understanding the chemistry behind DDAH catalysis and regulation, we will develop new biochemical tools with therapeutic potential, and will learn more about the role that DDAH plays in cancer biology and in the general response to oxidative and nitrosative stress. Relevance to Public Health: This application studies a key control point for the production of nitric oxide, a reactive chemical that can cause significant health problems such as promoting tumor growth in certain cancers and causing brain damage during stroke if it is not properly regulated. An understanding of the chemistry behind how this control valve works will allow us to understand how humans react to stressful physiological conditions, and will give us new biochemical tools that can be later developed into novel therapeutics.