Quantitative, High-throughput Mechanistic Enzymology

定量、高通量机械酶学

基本信息

批准号：
10013223
负责人：
Polly Morrell Fordyce
金额：
$ 56.95万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-01-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10013223
关键词：
Active Sites Address Affinity Alcohol dehydrogenase Alkaline Phosphatase Architecture Bacillus stearothermophilus Binding Biochemical Reaction Biological Biological Assay Biological Models Biology Catalysis Catalytic Domain Chemicals Complex Data Devices Distal Elements Engineering Enzymatic Biochemistry Enzyme Kinetics Enzyme Stability Enzymes Evolution Future Industry Investigation Isotopes Kinetics Ligand Binding Ligands Maps Measurement Medicine Metabolic Microfluidics Modeling Molecular Conformation Mutation Mutation Analysis Orthologous Gene Phylogenetic Analysis Positioning Attribute Property Protein Dynamics Proteins Reaction Recombinants Research Research Personnel Sampling Specificity Structure System Techniques Temperature Testing Thermodynamics Time Variant analog assay development base catalyst cost design direct application fitness in vivo inhibitor/antagonist insight interest member molecular dynamics mutant role model technological innovation thymidylate synthase-dihydrofolate reductase tool

项目摘要

Project Summary Enzymes are the primary catalysts of biological transformations and have enormous value in medicine and industry. While decades of research have established that active site residues are essential for catalysis, we do not yet understand in detail the contributions of residues beyond the active site to efficiency and specificity. Of course a folded enzyme is required for catalysis, but beyond folding there is a complex functional interplay of residues throughout an enzyme: allosteric ligand binding and remote mutations alter and enhance catalysis, active site residues coevolve with remote residues and regions, and distal mutations arise in screens and selections for enzymes with enhanced functional properties. Given this inherent complexity, our central premise is that we need tools that extend the power of traditional mechanistic enzymology to systematically investigate residues throughout the entire protein and their interconnectivity. Our central technological innovation delivers the needed tools: High-throughput Microfluidics for Enzyme Kinetics (HT-MEK) and Stability (HT-MES) expresses, purifies, and quantitatively assays 1200 enzyme variants in parallel, rapidly and inexpensively, yielding accurate kinetic and thermodynamic constants for many substrates and ligands over many conditions. With these measurements we will map functional regions and linkages throughout proteins, allowing enzymology to address previously inaccessible challenges in mechanism, evolution, and biology. We first apply these tools to the Alkaline Phosphatase (AP) superfamily member E. meningoseptica PafA, leveraging extensive prior structural, mechanistic, and phylogenetic insights to guide assay development and test previously inaccessible models to deepen our understanding of enzyme catalysis. We will systematically and quantitatively determine kinetic parameters for cognate and promiscuous PafA substrates and affinities for ground and transition-state PafA inhibitors, and we will do so for multiple mutations of every PafA residue; these measurements will provide a comprehensive map of enzyme regions that contribute to specific components of catalytic function. Next, we will use multi-mutant cycles to determine the energetic and functional linkage of these regions to active site residues and specific catalytic features, as well as the connections within and between these regions. Extension to multiple AP superfamily members across evolutionary distances will identify the range and limits of generality of functional maps and identify the alterations that rewire functional connectivity. Expanding this approach to other targets, some of which are explored herein, will address fundamental and practical problems of broad interest. This carefully-reasoned, stepwise approach will usher in a new era of enzymology, in which the acquisition of multidimensional functional maps of enzymes addresses new questions in mechanism, evolution, and biology, and in which enzymology impacts biomedicine and engineering in new ways.

项目摘要酶是生物转化的主要催化剂，在医学和行业。虽然数十年的研究已经确定活跃的现场残基对于催化至关重要，但我们确实如此尚未详细了解活跃部位以外的残留物对效率和特异性的贡献。的催化需要折叠的酶整个酶的残留物：变构配体结合和远程突变改变并增强催化，活性位点残基与远程残基和区域相结合，屏幕和远端突变发生在屏幕和具有增强功能特性的酶的选择。鉴于这种固有的复杂性，我们的中心前提是我们需要扩展传统机械酶学的力量来系统研究的工具整个蛋白质及其相互连接的残留物。我们的中心技术创新提供所需的工具：用于酶动力学的高通量微流体（HT-MEK）和稳定性（HT-MES）表达，净化和定量测定1200个并行，快速和廉价的酶变体在许多条件下，为许多底物和配体产生精确的动力学和热力学常数。通过这些测量值，我们将绘制整个蛋白质的功能区域和联系，从而允许酶学解决以前在机制，进化和生物学方面的挑战。我们首先将这些工具应用于碱性磷酸酶（AP）超家族成员E. Meningoseptica Pafa，利用广泛的先前结构，机械和系统发育见解来指导测定开发和测试以前无法访问的模型，以加深我们对酶催化的理解。我们将系统地并定量确定同源和混杂PAFA底物的动力学参数以及地面和过渡态PAFA抑制剂，我们将为每个PAFA残基的多个突变做到这一点；这些测量将为酶区域提供全面的地图，该酶区域有助于特定组成部分催化功能。接下来，我们将使用多突变循环来确定这些循环的能量和功能联系有效现场残留物和特定催化特征的区域，以及内部和之间的连接这些地区。跨进化距离扩展到多个AP超家族成员将确定功能图的一般性的范围和限制，并确定重新连接连接的变化。将这种方法扩展到其他目标，其中一些目标将在此探讨，将解决基本的问题广泛关注的实际问题。这种精心审查的逐步方法将迎来一个新时代酶学，在其中获取酶的多维功能图解决了新问题在机制，进化和生物学上，酶学影响了新的生物医学和工程方式。