Role of Binding Determinants in Enzyme Catalysis

结合决定因素在酶催化中的作用

• 批准号: RGPIN-2022-04282
• 负责人: Bearne, Stephen
• 金额: $ 4.88万
• 依托单位: Dalhousie University
• 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=744250
• 关键词: Role; Binding; Determinants; Enzyme; Catalysis

Enzymes are proteins that accelerate (catalyze) biological reactions. Our research focuses on understanding how catalysis arises from the interactions that occur between enzymes and the substrates they act on. This knowledge is essential to understand how enzymes work, to engineer new enzyme catalysts, and to design enzyme inhibitors (drugs/herbicides). Primarily, we study mandelate racemase (MR), which catalyzes the interconversion of D- and L-mandelic acid and serves as a paradigm for understanding how enzymes catalyze unfavorable reactions (i.e., C-H bond cleavage). Racemases and epimerases are of great interest because of their utility for the preparation of D-amino acids, rare sugars, and other value-added compounds. This proposal extends our research on enzymes through 4 themes. Theme [1] focuses on labelling catalytic acid-base residues of a racemase with NMR-active nuclei to distinguish their protonation states. These studies will furnish direct evidence of how substrate-binding alters the pKa of the acid-base catalysts at the active sites of racemases. This knowledge aids the rational design of novel enzyme catalysts. Theme [2] elaborates two enzyme inhibition strategies we developed: inhibition of enzymes acting on carbon acid substrates by boronic acids and the inhibition of racemases by substrate-product analogues (SPAs). We will examine thermodynamic and structural aspects of inhibition of MR by boronic acids to clarify how boronic acid-based inhibitors interact with such enzymes. The results will be of interest to pharma since the boronic acid group affords a point of molecular recognition on drugs and exhibits low toxicity. We will synthesize an SPA to obtain structural information on a non-ribosomal peptide synthetase (NRPS), which will inform the re-engineering of NRPSs to produce novel antibiotics. Theme [3] involves fully characterizing the kinetic and oligomerization properties of a "unidirectional" amino acid racemase. While some racemases may appear to be "unidirectional" under specific conditions, it is unlikely that they are truly unidirectional. We will develop a framework for understanding this behaviour based on the kinetic parameters and/or oligomerization properties of these enzymes. Theme [4] focuses on developing an activity-based protein profiling reagent for the detection of specific active-site architectures among all the proteins (proteome) in an organism. We have developed a reactive probe that will selectively label a specific architecture, thereby permitting identification of potential targets for drug development. This research program offers a multidisciplinary, respectful, collaborative, and inclusive training environment that will benefit trainees at all levels. Trainees develop skills in enzyme kinetics, protein chemistry, physical organic laboratory techniques, organic synthesis, microbiology, and molecular biology - assets for anyone pursuing a career in pharma, biotechnology, or academia.

酶是加速(催化)生物反应的蛋白质。我们的研究重点是了解酶和它们作用的底物之间的相互作用是如何产生催化作用的。这些知识对于了解酶的工作原理、设计新的酶催化剂和设计酶抑制剂(药物/除草剂)至关重要。首先，我们研究扁桃酸酯消旋酶(MR)，它催化D-和L-扁桃酸的相互转化，并作为理解酶如何催化不利反应(即C-H键断裂)的范例。消旋酶和外消旋异构酶因其在制备D-氨基酸、稀有糖和其他增值化合物方面的应用而引起人们的极大兴趣。这项建议通过4个主题扩展了我们对酶的研究。主题[1]的重点是用核磁共振活性核标记外消旋酶的催化酸碱残基，以区分它们的质子化状态。这些研究将为底物结合如何改变外消旋酶活性部位的酸碱催化剂的pKa提供直接证据。这些知识有助于新型酶催化剂的合理设计。主题[2]阐述了我们开发的两种酶抑制策略：用硼酸抑制作用于碳酸底物上的酶和用底物产物类似物(SPA)抑制外消旋酶。我们将研究硼酸对MR抑制的热力学和结构方面，以阐明基于硼酸的抑制剂如何与这些酶相互作用。这一结果将引起制药商的兴趣，因为硼酸基团在药物上提供了一个分子识别点，并且表现出低毒性。我们将合成一种SPA来获得非核糖体肽合成酶(NRPS)的结构信息，这将为NRPS的重组提供信息，以生产新的抗生素。主题[3]充分描述了“单向”氨基酸外消旋酶的动力学和寡聚性质。虽然一些外消旋酶在特定条件下可能看起来是“单向的”，但它们不太可能是真正的单向的。我们将根据这些酶的动力学参数和/或寡聚特性来开发一个框架来理解这一行为。Theme[4]的重点是开发一种基于活性的蛋白质图谱试剂，用于检测生物体中所有蛋白质(蛋白质组)中特定的活性部位结构。我们已经开发了一种反应性探针，它将选择性地标记特定的体系结构，从而允许识别潜在的药物开发目标。这一研究计划提供了一个多学科、尊重、协作和包容的培训环境，将使所有级别的受训者受益。受训者培养酶动力学、蛋白质化学、物理有机实验室技术、有机合成、微生物学和分子生物学方面的技能-这些技能是任何在制药、生物技术或学术界寻求职业生涯的人的资产。