Dihydropteridine Reductase: Mechanism of Enzyme Action

描述(由申请人提供)：酶-底物相互作用长期以来被认为是生物化学中结构互补性的极端表现。酶反应可以分为两个主要方面。第一个涉及由扩散控制的相遇复合体的形成，第二个涉及反应化学所决定的适当的酶结构域的结构和动态排列。PIS研究计划的长期目标是研究酶-小分子(抑制剂、底物或辅因子)的相互作用，以揭示其反应的结构和动力学方面。这项建议旨在研究DHPR中涉及的氢化物转移机理。脱氢蝶呤还原酶(DHPR)催化NADH介导的二氢生物蝶呤(QBH2)还原生成四氢生物蝶呤(BH4)。BH4是一种重要的辅因子，缺乏BH4会导致大脑中儿茶酚胺和5-羟色胺神经递质前体的枯竭。当发现一种与BH4循环缺陷相关的新形式的高苯丙氨酸血症(非典型苯丙酮尿症)时，对DHPR的调节变得令人感兴趣。鉴于其他新出现的功能，人们越来越需要完全了解DHPR是如何工作的。研究的总体目标是了解DHPR与辅因子、底物或抑制剂结合时发生的结构变化。使用先进的荧光和振动技术，我们将识别催化重要的键，并检测可能伴随结合和催化而来的主要蛋白质构象变化。我们还将使用基于荧光的温度跳跃研究来研究结合和催化事件的动力学方面。这些结果将与DHPR已解决的X射线晶体结构相结合，以深入了解酶的作用机制。特别是，我们想要解决辅因子和保守的半胱氨酸残基的机械作用。由于DHPR属于具有重要生物学意义的短链脱氢酶/还原酶(SDRs)超家族，因此这些研究结果有望为SDRs的研究提供一些有用的机制细节。特别提款权具有多种功能。有些是很好的药理靶点，而另一些则在控制激素受体配体的细胞可利用性方面发挥着重要作用。也有一些在生长因子或营养合成中起重要作用的物质。在分子水平上了解DHPR的酶学特性，将有助于实现药物设计、酶调节和酶工程的巨大潜力。

DESCRIPTION (provided by applicant): Enzyme-substrate interactions have long been recognized as representing an extreme expression of structural complementarities in biological chemistry. An enzymatic reaction can be broken down into two main aspects. The first involves the diffusion-controlled formation of the encounter complex, and the second involves the appropriate structural and dynamical arrangements of the enzyme domains as dictated by the reaction chemistry. It is the long-term goal of the PIs research program to investigate enzyme-small molecule (inhibitor, substrate, or cofactor) interactions in order to unravel the structural and dynamical aspects of its reaction. This proposal aims to study the hydride transfer mechanism involved in DHPR. Dehydropteridine reductase (DHPR) catalyses the NADH-mediated reduction of quinonoid dihydrobiopterin (qBH2) to yield tetrahydrobiopterin (BH4). BH4 functions as an essential cofactor, and its absence leads to depletion in the brain of precursors of catecholamine and serotonin neurotransmitters. Regulation of DHPR became of interest when a new form of hyperphenylalanineanemia (atypical phenylketonuria) associated with a defect in BH4 recycling was discovered. In the light of other new emerging functions, there is a growing need to completely understand how DHPR works. The overall objective of the investigation is to understand the structural changes that occur when DHPR binds cofactor, substrate or inhibitor. Using advanced fluorescence and vibrational techniques, we will identify catalytically important bonds as well as detect major protein conformational changes that may accompany binding and catalysis. We will also look at the dynamical aspects of the binding and catalytic events using fluorescence-based temperature-jump studies. The results will be married to the solved X-ray crystal structures of DHPR to gain insights into the mechanism of enzyme action. In particular, we would like to address the mechanistic role of the cofactor and the conserved Cys residue. Since DHPR belongs to a superfamily of biologically important short-chain dehydrogenases/reductases (SDRs), it is hoped that the gathered results could provide some mechanistic details useful for studying SDRs. SDRs have diverse functions. Some are good pharmacological targets while others are important in controlling the cellular availability of a hormone receptor ligand. There are also those that have essential role in growth factors or nutrient synthesis. Understanding the enzymology of DHPR at the molecular level will play a part in realizing the untold potential for drug design, enzyme regulation, and enzyme engineering.