Role of Binding Determinants in Enzyme Catalysis

酶是加速(催化)生物反应的蛋白质。我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CH键断裂)的范例。以前，我们发现MR被底物-产物类似物抑制，并开发了一种通用的设计策略来抑制辅因子无关的消旋酶。本提案通过四个主题扩展了这一研究方案。主题[1]提出了这样的假设，即底物结合调节MR的Brnsted酸碱催化剂Lys 166的环境(因此调节其PKA)。将在位置166引入光笼-15N-Lys。去保护后，15N核磁共振波谱将被用来观察底物类似物的结合如何改变15N化学位移，从而揭示配体结合如何改变外消旋酶的Brnsted酸碱催化剂的环境。Theme[2]的重点是改变MR的疏水空腔以改变其底物专一性。我们的假设是，相邻亚基之间的交叉环是MR和相关酶D-酒石酸脱水酶(TARD)之间底物专一性的主要决定因素。这个环的末端(Leu93)将发生突变，以确定对MR催化和寡聚态的影响。末端的Lys增强了MR对D-酒石酸盐的结合，这将成为将MR工程转化为TARD的起点。这些研究将提供将底物专一性构建到疏水性口袋中的“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(S)。初步研究表明，MR可以调节结合金属离子的有效电荷。通过突变来削弱MR与金属的相互作用将被用来检验这一假说。还将探索TARD对金属离子的依赖性，以发现金属离子是否对催化是必要的。Theme[4]的重点是开发一种基于活性的蛋白质图谱分析试剂，以确定蛋白质组中的活性部位结构。一种基于甲酰基磷酸的试剂将被用来鉴定含有靠近阳离子中心的亲核中心的酶。这种方法应该能够识别蛋白质组中具有共同反应基序的酶家族，这些反应基序可以作为抑制剂/药物开发的靶点。在这个多学科的环境中，受训人员为任何对制药、生物技术或学术界感兴趣的人开发有机合成、蛋白质化学、酶动力学、微生物学和分子生物学资产方面的技能。**

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., CH 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 -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.**