Structural and functional studies of allosteric regulation of metabolic enzymes

自然界进化出了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、最快速、最有效的方法来感知和改变小分子的浓度。在变构过程中，一个大分子的功能部位受到与另一个远端部位结合的调节分子的影响。这一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，通常被称为效应器，与参与功能步骤的分子有很大不同。这个由NSERC支持的计划的长期目标是识别变构调节的酶及其效应器，阐明起作用的分子作用力和机制，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)确定法尼基焦磷酸合成酶的变构效应并探索该酶的构象转变；ii)表征人甲伐他酸激酶的结构并确定该酶的变构抑制剂；iii)阐明单价阳离子调节核糖激酶的分子和细胞机制。拟议的研究将涉及一系列方法。感兴趣的酶将在基因操纵的细菌细胞中生产，并通过使用层析技术进行纯化。通过X射线结晶学显示酶的分子结构，并通过体外测试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重要的生物学过程中都发挥着关键作用，但它仍然是一个被低估的研究课题。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)，但大多数变构调节的酶及其自然效应尚未确定。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。这一由NSERC资助的研究项目的成果将有助于填补这些空白，并为推进系统生物学和分子生物物理学领域做出贡献。更深入地了解变构过程将在药物发现和蛋白质工程等应用领域具有重要意义。

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.