Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

被引：116

作者：

Markin, C. J. ^{[1
]}

Mokhtari, D. A. ^{[1
]}

Sunden, F. ^{[1
]}

Appel, M. J. ^{[1
]}

Akiva, E. ^{[2
]}

Longwell, S. A. ^{[3
]}

Sabatti, C. ^{[4
,5
]}

Herschlag, D. ^{[1
,6
,7
]}

Fordyce, P. M. ^{[3
,7
,8
,9
]}

机构：

[1] Stanford Univ, Dept Biochem, Stanford, CA 94305 USA

[2] Univ Calif San Francisco, Dept Bioengn & Therapeut Sci, San Francisco, CA 94158 USA

[3] Stanford Univ, Dept Bioengn, Stanford, CA 94305 USA

[4] Stanford Univ, Dept Biomed Data Sci, Stanford, CA 94305 USA

[5] Stanford Univ, Dept Stat, Stanford, CA 94305 USA

[6] Stanford Univ, Dept Chem Engn, Stanford, CA 94305 USA

[7] Stanford Univ, ChEM H Inst, Stanford, CA 94305 USA

[8] Stanford Univ, Dept Genet, Stanford, CA 94305 USA

[9] Chan Zuckerberg Biohub, San Francisco, CA 94110 USA

来源：

SCIENCE | 2021年 / 373卷 / 6553期

基金：

加拿大健康研究院;

关键词：

ALPHA-LYTIC PROTEASE; ALKALINE-PHOSPHATASE; BINDING-ENERGY; TRANSCRIPTION-FACTOR; ACTIVE-SITE; COMPARATIVE ENZYMOLOGY; INORGANIC-PHOSPHATE; ALLOSTERIC SITES; GROUND-STATE; CATALYSIS;

D O I：

10.1126/science.abf8761

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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.

引用

页码：411 / +

页数：161

共 75 条

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