Quantitative, High-throughput Mechanistic Enzymology

项目摘要 酶是生物转化的主要催化剂，在医学和生物医学领域具有巨大的价值。 行业虽然几十年的研究已经确定，活性位点残基是催化必不可少的，我们做的， 尚未详细了解活性位点以外的残基对效率和特异性的贡献。的 当然，一个折叠的酶是催化所必需的，但除了折叠，还有一个复杂的功能相互作用， 整个酶中的残基：变构配体结合和远程突变改变并增强催化， 活性位点残基与远端残基和区域共同进化，并且远端突变在筛选中出现， 选择具有增强功能特性的酶。鉴于这种内在的复杂性，我们的中心前提 我们需要一些工具来扩展传统的机械酶学的力量， 整个蛋白质中的残基及其相互连接。我们的核心技术创新 所需的工具：高通量微流体酶动力学（HT-MEK）和稳定性（HT-MES） 平行、快速且廉价地表达、纯化和定量测定1200种酶变体， 在许多条件下产生许多底物和配体的精确动力学和热力学常数。 通过这些测量，我们将绘制整个蛋白质的功能区域和连接， 以解决以前在机制，进化和生物学方面无法实现的挑战。 我们首先将这些工具应用于碱性磷酸酶（AP）超家族成员E。脑膜炎败血症PafA， 利用广泛的先前结构、机制和系统发育见解来指导检测开发， 测试以前无法实现的模型，以加深我们对酶催化的理解。我们将系统地 并定量测定同源和混杂的PafA底物的动力学参数以及 基态和过渡态PafA抑制剂，我们将对每个PafA残基的多个突变这样做;这些 测量将提供酶区域的全面地图，这些酶区域有助于特定的组分， 催化功能。接下来，我们将使用多突变周期来确定这些基因的能量和功能联系。 区域到活性位点残基和特定催化特征，以及内部和之间的连接 这些地区。跨越进化距离扩展到多个AP超家族成员将识别 功能图的一般性范围和限制，并确定重新连接功能连接的变更。 将这一方法扩展到其他目标，其中一些将在本文中探讨， 广泛关注的实际问题。这种精心推理的逐步方法将迎来一个新的时代， 酶学，其中酶的多维功能图的获取解决了新问题 在机制，进化和生物学，其中酶学影响新的生物医学和工程学 的方式

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.