4.8 Article

Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Journal

SCIENCE
Volume 373, Issue 6553, Pages 411-+

Publisher

AMER ASSOC ADVANCEMENT SCIENCE
DOI: 10.1126/science.abf8761

Keywords

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Funding

  1. NIH [RO1, GM064798, GM0595]
  2. Joint Initiative for Metrology in Biology (JIMB) seed grant, a Stanford Bio-X Interdisciplinary Initiative Seed Grant
  3. Ono Pharma Foundation Breakthrough Innovation Prize
  4. Gordon and Betty Moore Foundation [8415]
  5. Alfred P. Sloan Foundation fellowship
  6. Canadian Institutes of Health Research (CIHR) Postdoctoral Fellowship
  7. Stanford Medical Scientist Training Program
  8. Stanford Interdisciplinary Graduate Fellowship
  9. US Department of Energy (DOE) Office of Science User Facility [DEAC02-05CH11231]

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A high-throughput microfluidic enzyme kinetics platform, HT-MEK, was studied for simultaneous expression, purification, and characterization of over 1500 enzyme variants. Through more than 670,000 reactions on 1036 mutants of alkaline phosphatase PafA, over 5000 kinetic and physical constants were determined, uncovering underlying enzyme architecture.
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.

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