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: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=615487
• 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。这些知识对于理解酶的工作原理、设计新的酶催化剂和设计酶抑制剂（药物/除草剂）至关重要。我主要研究扁桃酸消旋酶（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键切割）的范例。在此之前，我们发现MR被底物产物类似物抑制，并开发了一种抑制辅因子无关外消旋酶的一般设计策略。本提案通过四个主题扩展了这一研究计划。主题[1]提出了底物结合调节Brønsted酸碱催化剂Lys 166（及其pKa）的环境的假设。在166位置引入一个光笼化的ε-15N-Lys。脱保护后，15N NMR将观察底物类似物的结合如何改变15N的化学位移，从而表明配体结合如何改变Brønsted酸碱外消旋酶催化剂的环境。主题[2]侧重于改变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 -酒石酸脱水酶（ard）之间底物特异性的主要决定因素。该环的尖端（Leu 93）将发生突变，以确定对MR催化和低聚物状态的影响。末端的赖氨酸增强了MR与酒石酸d的结合，这将作为MR进入ard的起点。这些研究将为在疏水口袋中构建底物特异性提供“规则”。主题[3]探讨金属离子在烯醇化酶超家族酶中的作用。初步研究表明，磁共振调节了结合金属离子的有效电荷。通过诱变削弱MR-metal相互作用将用于验证这一假设。我们还将探讨TarD对金属离子的依赖性，以发现金属离子是否是催化所必需的。主题[4]的重点是开发一种基于活性的蛋白质分析试剂来识别蛋白质组中的活性位点结构。甲基酰基磷酸酯为基础的试剂将被用来鉴定酶含有亲核位点毗邻阳离子位点。这种方法应该允许鉴定蛋白质组中共享反应性基序的酶家族，这些基序可以作为抑制剂/药物开发的目标。在这个多学科的环境中，学员可以培养有机合成、蛋白质化学、酶动力学、微生物学和分子生物学方面的技能，这对任何有兴趣在制药、生物技术或学术界从事职业的人来说都是宝贵的财富。