美国斯坦福大学P. M. Fordyce和D. Herschlag团队合作取得一项新成果。经过不懈努力，他们通过高通量微流体酶动力学揭示了酶的功能结构。该研究于2021年7月23日发表于国际学术期刊《科学》杂志。

研究人员研发了HT-MEK（高通量微流体酶动力学），这是一个微流控平台，单次实验可实现对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，研究人员进行了超过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。

研究发现了错误折叠状态下的广泛动力学区室和独立的催化作用，揭示了与特定功能相关的残基空间连续区域。这些区域包括活性位点近端的残基，但延伸到酶表面，提供了现有方法无法获得的底层结构图。HT-MEK具有从揭示分子机制到医学、工程和设计等方面的广泛应用。

据了解，需要对酶进行系统且广泛的研究以揭示它们高催化效率的机制，从而解决当前医学和工程领域所面临的难题。

附：英文原文

Title: Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Author: C. J. Markin, D. A. Mokhtari, F. Sunden, M. J. Appel, E. Akiva, S. A. Longwell, C. Sabatti, D. Herschlag, P. M. Fordyce

Issue&Volume: 2021/07/23

Abstract: 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.

DOI: 10.1126/science.abf8761

Source: https://science.sciencemag.org/content/373/6553/eabf8761