Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

C J Markin; D A Mokhtari; F Sunden; M J Appel; E Akiva; S A Longwell; C Sabatti; D Herschlag; P M Fordyce

doi:10.1126/science.abf8761

Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Science. 2021 Jul 23;373(6553):eabf8761. doi: 10.1126/science.abf8761.

Authors

C J Markin^#¹, D A Mokhtari^#¹, F Sunden¹, M J Appel¹, E Akiva², S A Longwell³, C Sabatti^{4

5}, D Herschlag^{6

7

8}, P M Fordyce^{9

8

10

11}

Affiliations

¹ Department of Biochemistry, Stanford University, Stanford, CA 94305, USA.
² Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA 94158, USA.
³ Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
⁴ Department of Biomedical Data Science, Stanford University, Stanford, CA 94305, USA.
⁵ Department of Statistics, Stanford University, Stanford, CA 94305, USA.
⁶ Department of Biochemistry, Stanford University, Stanford, CA 94305, USA. herschla@stanford.edu pfordyce@stanford.edu.
⁷ Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA.
⁸ ChEM-H Institute, Stanford University, Stanford, CA 94305, USA.
⁹ Department of Bioengineering, Stanford University, Stanford, CA 94305, USA. herschla@stanford.edu pfordyce@stanford.edu.
¹⁰ Department of Genetics, Stanford University, Stanford, CA 94305, USA.
¹¹ Chan Zuckerberg Biohub; San Francisco, CA 94110, USA.

^# Contributed equally.

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.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Alkaline Phosphatase / antagonists & inhibitors
Alkaline Phosphatase / chemistry
Alkaline Phosphatase / genetics*
Alkaline Phosphatase / metabolism*
Biocatalysis
Catalytic Domain
Flavobacterium / enzymology
Hydrolysis
Kinetics
Microfluidics
Models, Molecular
Mutation
Oxygen / metabolism
Phosphates / metabolism
Protein Conformation
Protein Folding
Thermodynamics

Substances

Phosphates
Alkaline Phosphatase
Oxygen

Grants and funding

R01 GM064798/GM/NIGMS NIH HHS/United States