Dissecting the Conformational Dynamics-Modulated Enzyme Catalysis with Single-Molecule FRET

The Journal of Physical Chemistry. B
Shaowen WuWenning Wang

Abstract

Conformational changes of enzyme proteins are often coupled with a catalytic reaction and modulate the enzyme activity. Single-molecule technology is a powerful tool to study the mechanism of enzyme catalysis in these complicated cases. However, the chemical reaction cycles and conformational changes could not be monitored simultaneously in a single-molecule detection experiment, resulting in some unresolved key kinetic parameters. Here, we describe a method to extract all of the kinetic parameters from comprehensive single-molecule FRET (smFRET) measurements and model analysis. On the basis of the smFRET, we calculated the undetectable parameters by solving the rate equations of the kinetic model with the input of the smFRET-measured conformational state populations and state-transition rate constants. A case study of MalK2 ATPase demonstrates that this method could reveal the quantitative mechanism of the catalytic reaction of the enzyme as well as its coupled conformational dynamics. The strategy employed in this study could be widely applied to investigate the conformational fluctuation-coupled catalysis of other enzymes.

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Citations

May 7, 2020·Pflügers Archiv : European journal of physiology·Jean-Marc Jeckelmann, Bernhard Erni
Sep 29, 2019·Research in Microbiology·Rebecca MächtelCédric Orelle

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