Structural and functional studies of allosteric regulation of metabolic enzymes

代谢酶变构调节的结构和功能研究

• 批准号: RGPIN-2020-04281
• 负责人: Park, Jaeok
• 金额: $ 2.19万
• 依托单位: Memorial University of Newfoundland
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744095
• 关键词: Structural; functional; studies; allosteric; regulation

Nature has evolved many redundant and complementary mechanisms to regulate cellular functions. Among these, allostery provides the most direct, rapid, and efficient means to sense and alter the concentration of small molecules. In an allosteric process, the functional site of a macromolecule is affected by a regulatory molecule binding to another, distally located site. This process is advantageous in controlling macromolecular function because it allows the regulatory molecules, commonly referred to as effectors, to differ widely from the molecules involved in the functional step. The long-term goals of this NSERC supported program are to identify allosterically regulated enzymes and their effectors, elucidate the molecular forces and mechanisms at work, and explore the significance of these mechanisms in metabolic pathways. I am particularly interested in the enzymes involved in two distinct processes, namely, isoprenoid biosynthesis (produces essential fatty metabolites) and monosaccharide phosphorylation (the first step in simple sugar metabolism). The short-term objectives for the next five years are: I) to identify allosteric effectors of farnesyl pyrophosphate synthase and explore the enzyme's conformational transition; II) to characterize the structure of human mevalonate kinase and identify allosteric inhibitors of the enzyme; and III) to elucidate the molecular and cellular mechanisms of ribokinase regulation by monovalent cations. The proposed studies will involve an array of methods. The enzymes of interest will be produced in genetically manipulated bacterial cells and purified by employing chromatographic techniques. Visualization of the enzymes' molecular structures by X-ray crystallography and assessment of their functional activities by in vitro assays will be the primary means of achieving the research objectives. Other biophysical methods that can indirectly characterize molecular interactions, such as isothermal titration calorimetry, will play a role in providing complementary data. Computational molecular simulations and tissue culture work will be carried out for some aspects of the proposed studies. Despite the mounting evidence that allosteric regulation plays a critical role in every essential biological process, it is still an underappreciated research subject. Many of the best-known examples of allosteric regulation have been discovered decades ago (e.g., hemoglobin), with the majority of allosterically regulated enzymes and their natural effectors yet to be identified. Moreover, the molecular mechanisms by which the fundamental physical properties of macromolecules underpin allostery are still poorly understood. The outcomes of this NSERC-funded research program will help fill these gaps and contribute to advancing the fields of systems biology and molecular biophysics. A deeper understanding of allosteric processes will have powerful implications in applied areas, such as drug discovery and protein engineering.

自然界已经进化出许多冗余和互补的机制来调节细胞功能。其中，变构提供了最直接、快速和有效的手段来感知和改变小分子的浓度。在变构过程中，大分子的功能位点受到与另一个远端位点结合的调节分子的影响。这个过程在控制大分子功能方面是有利的，因为它允许调节分子（通常称为效应物）与功能步骤中涉及的分子有很大的不同。这个NSERC支持计划的长期目标是识别变构调节酶及其效应物，阐明工作中的分子力和机制，并探索这些机制在代谢途径中的意义。我特别感兴趣的是参与两个不同过程的酶，即类异戊二烯生物合成（产生必需的脂肪代谢产物）和单糖磷酸化（简单糖代谢的第一步）。未来五年的短期目标是：I）鉴定法呢基焦磷酸合酶的变构效应物并探索该酶的构象转变; II）表征人甲羟戊酸激酶的结构并鉴定该酶的变构抑制剂;和III）阐明单价阳离子对核糖激酶调节的分子和细胞机制。拟议的研究将涉及一系列方法。感兴趣的酶将在基因操作的细菌细胞中产生，并通过采用色谱技术纯化。通过X射线晶体学可视化酶的分子结构并通过体外试验评估其功能活性将是实现研究目标的主要手段。其他可以间接表征分子相互作用的生物物理方法，如等温滴定量热法，将在提供补充数据方面发挥作用。将对拟议研究的某些方面进行计算分子模拟和组织培养工作。尽管越来越多的证据表明，变构调节在每一个基本的生物过程中发挥着关键作用，它仍然是一个未得到充分重视的研究课题。许多最著名的变构调节的例子已经在几十年前被发现（例如，血红蛋白），其中大多数变构调节的酶及其天然效应物尚待鉴定。此外，大分子的基本物理性质支持变构的分子机制仍然知之甚少。这个NSERC资助的研究项目的成果将有助于填补这些空白，并有助于推进系统生物学和分子生物物理学领域。对变构过程的深入理解将在药物发现和蛋白质工程等应用领域产生重大影响。