Recent experimental results have shown that enzymes can diffuse faster when they are in the presence of their reactants (substrate). This faster diffusion has been termed enhanced diffusion. ...Fluorescence correlation spectroscopy (FCS), which has been employed as the only method to make these measurements, relies on analyzing the fluctuations in fluorescence intensity to measure the diffusion coefficient of particles. Recently, artifacts in FCS measurements due to its sensitivity to environmental conditions have been evaluated, calling prior enhanced diffusion results into question. It behooves us to adopt complementary and direct methods to measure the mobility of enzymes. Herein, we use a technique of direct single molecule imaging to observe the diffusion of individual enzymes in solution. This technique is less sensitive to intensity fluctuations and deduces the diffusion coefficient directly based on the trajectory of the enzyme. Our measurements recapitulate that enzyme diffusion is enhanced in the presence of its substrate and find that the relative increase in diffusion of a single enzyme is even higher than those previously reported using FCS. We also use this complementary method to test if the total enzyme concentration affects the relative increase in diffusion and if the enzyme oligomerization state changes during its catalytic turnover. We find that the diffusion increase is independent of the total concentration of enzymes and the presence of substrate does not change the oligomerization state of enzymes.
Recent experimental results have shown that active enzymes can diffuse faster when they are in the presence of their substrates. Fluorescence correlation spectroscopy (FCS), which relies on analyzing ...the fluctuations in fluorescence intensity signal to measure the diffusion coefficient of particles, has typically been employed in most of the prior studies. However, flaws in the FCS method, due to its high sensitivity to the environment, have recently been evaluated, calling the prior diffusion results into question. It behooves us to adopt complimentary and direct methods to measure the mobility of enzymes in solution. Herein, we use a novel technique of direct single-molecule imaging to observe the diffusion of single enzymes. This technique is less sensitive to intensity fluctuations and gives the diffusion coefficient directly based on the trajectory of the enzymes. Our measurements recapitulate that enzyme diffusion is enhanced in the presence of its substrate and find that the relative increase in diffusion of a single enzyme is even higher than those previously reported using FCS. We also use this complementary method to test if the total enzyme concentration affects the relative increase in diffusion and if enzyme oligomerization state changes during catalytic turnover. We find that the diffusion increase is independent of the total background concentration of enzyme and the catalysis of substrate does not change the oligomerization state of enzymes.
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