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bioRxiv · 10.1101/2020.11.24.383182

Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Markin, C. J.·Mokhtari, D. A.·Sunden, F.·Appel, M. J.·Akiva, E.·Longwell, S.·Sabatti, C.·Herschlag, D.·Fordyce, P. M.
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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, a microfluidic platform for high-throughput expression, purification, and characterization of >1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA, we performed >670,000 reactions to determine >5000 kinetic and physical constants for multiple substrates and inhibitors. These constants allowed us to uncover extensive kinetic partitioning to a misfolded state and isolate catalytic effects, revealing spatially contiguous "regions" of residues linked to particular aspects of function. These regions included active-site proximal residues but also extended to the enzyme surface, providing a map of underlying architecture that could not be derived from existing approaches. HT-MEK, using direct and coupled fluorescent assays, has future applications to a wide variety of problems ranging from understanding molecular mechanisms to medicine to engineering and design. One Sentence SummaryHT-MEK, a microfluidic platform for high-throughput, quantitative biochemistry, reveals enzyme architectures shaping function.

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Markin, C. J., Mokhtari, D. A., Sunden, F., Appel, M. J., Akiva, E., Longwell, S., Sabatti, C., Herschlag, D., Fordyce, P. M.. 2020-11-25. Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics. https://doi.org/10.1101/2020.11.24.383182

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