Resolution of Submillisecond Kinetics of Multiple Reaction Pathways for Lactate Dehydrogenase

Biophysical Journal
Michael J ReddishR Brian Dyer

Abstract

Enzymes are known to exhibit conformational flexibility. An important consequence of this flexibility is that the same enzyme reaction can occur via multiple reaction pathways on a reaction landscape. A model enzyme for the study of reaction landscapes is lactate dehydrogenase. We have previously used temperature-jump (T-jump) methods to demonstrate that the reaction landscape of lactate dehydrogenase branches at multiple points creating pathways with varied reactivity. A limitation of this previous work is that the T-jump method makes only small perturbations to equilibrium and may not report conclusively on all steps in a reaction. Therefore, interpreting T-jump results of lactate dehydrogenase kinetics has required extensive computational modeling work. Rapid mixing methods offer a complementary approach that can access large perturbations from equilibrium; however, traditional enzyme mixing methods like stopped-flow do not allow for the observation of fast protein dynamics. In this report, we apply a microfluidic rapid mixing device with a mixing time of <100 μs that allows us to study these fast dynamics and the catalytic redox step of the enzyme reaction. Additionally, we report UV absorbance and emission T-jump results w...Continue Reading

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Citations

Apr 27, 2019·Toxicology and Industrial Health·Huichao ZhaoZhihong Zhang
Aug 29, 2018·MedChemComm·Brooke A Andrews, R Brian Dyer
Mar 30, 2018·The Journal of Physical Chemistry. B·Juan M Hernández-Meza, José G Sampedro
Oct 24, 2019·The Journal of Physical Chemistry. B·Tsuyoshi EgawaRobert Callender

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