Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), we performed more than 670,000 reactions and determined more than 5000 kinetic and physical constants for multiple substrates and inhibitors. We uncovered extensive kinetic partitioning to a misfolded state and isolated catalytic effects, revealing spatially contiguous regions of residues linked to particular aspects of function. Regions included active-site proximal residues but extended to the enzyme surface, providing a map of underlying architecture not possible to derive from existing approaches. HT-MEK has applications that range from understanding molecular mechanisms to medicine, engineering, and design.

需要对酶进行系统和广泛的研究，以了解它们的非凡效率并应对当前医学和工程领域的挑战。我们展示了 HT-MEK（高通量微流控酶动力学），这是一个微流控平台，用于对每个实验 1500 多种酶变体进行高通量表达、纯化和表征。对于碱性磷酸酶 PafA（黄杆菌的磷酸不可抑制碱性磷酸酶）的 1036 个突变体，我们进行了超过 670,000 次反应，并确定了多种底物和抑制剂的 5000 多个动力学和物理常数。我们发现了对错误折叠状态的广泛动力学分区和孤立的催化效应，揭示了与特定功能方面相关的残基的空间连续区域。区域包括活性位点近端残基，但延伸到酶表面，提供了不可能从现有方法中获得的底层结构图。HT-MEK 的应用范围从理解分子机制到医学、工程和设计。