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-2016-05083
• 负责人: Bearne, Stephen
• 金额: $ 2.77万
• 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: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=644866
• 关键词: Role; Binding; Determinants; Enzyme; Catalysis

Enzymes are proteins that accelerate (catalyze) biological reactions. My 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, I 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). Previously, we discovered that MR is inhibited by substrate-product analogues and developed a general design strategy for inhibiting cofactor-independent racemases. The present proposal extends this research program through four themes. Theme [1] addresses the hypothesis that substrate binding modulates the environment of the Brønsted acid-base catalyst Lys 166 of MR (and hence its pKa). A photocaged ε-15N-Lys will be introduced at position 166. After deprotection, 15N NMR spectroscopy will be used to observe how the binding of substrate analogues changes the 15N chemical shift, thereby indicating how ligand binding alters the environment of the Brønsted acid-base catalysts of racemases. Theme [2] focuses on altering the hydrophobic cavity of MR to change its substrate specificity. Our hypothesis is that an interdigitating loop between adjacent subunits is a prime determinant of substrate specificity between MR and the related enzyme D-tartrate dehydratase (TarD). The tip of this loop (Leu 93) will be mutated to determine the effect on MR catalysis and oligomeric state. Lys at the tip enhances the binding of D-tartrate by MR and this will serve as a starting point for engineering MR into TarD. These studies will provide the “rules” for building substrate specificity into a hydrophobic pocket. Theme [3] explores the role(s) of metal ions in enolase superfamily enzymes. Preliminary studies suggest that MR modulates the effective charge of the bound metal ion. Weakening the MR-metal interactions through mutagenesis will be used to test this hypothesis. The metal ion dependence of TarD will also be explored to discover if the metal ion is essential for catalysis. Theme [4] focuses on developing an activity-based protein profiling agent to identify active site architectures in proteomes. A methyl acyl phosphate-based reagent will be used to identify enzymes containing a nucleophilic site adjacent to a cationic site. This approach should permit identification of families of enzymes in proteomes that share reactive motifs that could be targeted for inhibitor/drug development. Trainees in this multidisciplinary environment develop skills in organic synthesis, protein chemistry, enzyme kinetics, microbiology, and molecular biology – assets for anyone interested in pursuing careers in pharma, biotechnology, or academia.**

酶是加速（催化）生物反应的蛋白质。我的研究重点是了解催化是如何从酶和它们作用的底物之间发生的相互作用中产生的。这些知识对于了解酶如何工作，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，我研究扁桃酸消旋酶（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键裂解）。在此之前，我们发现MR被底物产物类似物抑制，并开发了一种抑制非辅因子依赖性消旋酶的通用设计策略。目前的建议通过四个主题扩展了这一研究计划。主题[1]提出了以下假设：底物结合调节MR的Brønsted酸碱催化剂Lys 166的环境（因此调节其pKa）。将在位置166处引入光笼化的ε-15 N-Lys。脱保护后，15 N NMR光谱将用于观察底物类似物的结合如何改变15 N化学位移，从而表明配体结合如何改变消旋酶的布朗斯特酸碱催化剂的环境。主题[2]关注改变MR的疏水腔以改变其底物特异性。我们的假设是相邻亚基之间的交错环是MR和相关酶D-酒石酸酯酶（TardD）之间底物特异性的主要决定因素。将突变该环的尖端（Leu 93）以确定对MR催化和寡聚状态的影响。尖端的赖氨酸增强了MR与D-酒石酸盐的结合，这将作为将MR工程化到Tar D中的起点。这些研究将为将底物特异性构建到疏水口袋中提供“规则”。主题[3]探讨了金属离子在烯醇化酶超家族酶中的作用。初步研究表明，MR调制的有效电荷的结合金属离子。通过诱变减弱MR-金属相互作用将用于检验该假设。还将探索TardD的金属离子依赖性，以发现金属离子是否对催化至关重要。主题[4]专注于开发一种基于活性的蛋白质分析试剂，以识别蛋白质组中的活性位点结构。基于酰基磷酸甲酯的试剂将用于鉴定含有与阳离子位点相邻的亲核位点的酶。这种方法应该允许识别蛋白质组中的酶家族，这些酶家族共享可用于抑制剂/药物开发的反应基序。在这个多学科的环境中，学员将培养有机合成，蛋白质化学，酶动力学，微生物学和分子生物学方面的技能-对于任何有兴趣从事制药，生物技术或学术界职业的人来说都是资产。