Resolution of Submillisecond Kinetics of Multiple Reaction Pathways for Lactate Dehydrogenase

Michael J. Reddish, Robert Callender, R. Brian Dyer

Research output: Contribution to journalArticle

3 Citations (Scopus)

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 with improved signal-to-noise ratio at fast times. The combination of mixing and T-jump results yields an unprecedented view of lactate dehydrogenase enzymology, confirming the timescale of substrate-induced conformational change and presence of multiple reaction pathways.

Original languageEnglish (US)
Pages (from-to)1852-1862
Number of pages11
JournalBiophysical Journal
Volume112
Issue number9
DOIs
StatePublished - May 9 2017

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L-Lactate Dehydrogenase
Temperature
Enzymes
Microfluidics
Signal-To-Noise Ratio
Oxidation-Reduction
Oxidoreductases
Observation
Equipment and Supplies
Proteins

ASJC Scopus subject areas

  • Biophysics

Cite this

Resolution of Submillisecond Kinetics of Multiple Reaction Pathways for Lactate Dehydrogenase. / Reddish, Michael J.; Callender, Robert; Dyer, R. Brian.

In: Biophysical Journal, Vol. 112, No. 9, 09.05.2017, p. 1852-1862.

Research output: Contribution to journalArticle

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