Resolution of Submillisecond Kinetics of Multiple Reaction Pathways for Lactate Dehydrogenase.

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 ?s 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.

SUBMITTER: Reddish MJ 

PROVIDER: S-EPMC5425397 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature