Master curve of boosted diffusion for 10 catalytic enzymes

Abstract

Molecular agitation more rapid than thermal Brownian motion is reported for cellular environments, motor proteins, synthetic molecular motors, enzymes, and common chemical reactions, yet that chemical activity coupled to molecular motion contrasts with generations of accumulated knowledge about diffusion at equilibrium. To test the limits of this idea, a critical testbed is the mobility of catalytically active enzymes. Sentiment is divided about the reality of enhanced enzyme diffusion, with evidence for and against. Here a master curve shows that the enzyme diffusion coefficient increases in proportion to the energy release rate—the product of Michaelis-Menten reaction rate and Gibbs free energy change (ΔG)—with a highly satisfactory correlation coefficient of 0.97. For 10 catalytic enzymes (urease, acetylcholinesterase, seven enzymes from the glucose cascade cycle, and one other), our measurements span from a roughly 40% enhanced diffusion coefficient at a high turnover rate and negativeΔGto no enhancement at a slow turnover rate and positiveΔG. Moreover, two independent measures of mobility show consistency, provided that one avoids undesirable fluorescence photophysics. The master curve presented here quantifies the limits of both ideas, that enzymes display enhanced diffusion and that they do not within instrumental resolution, and has possible implications for understanding enzyme mobility in cellular environments. The striking linear dependence of ΔGfor the exergonic enzymes (ΔG<0), together with the vanishing effect for endergonic enzyme (ΔG>0), are consistent with a physical picture in which the mechanism boosting the diffusion is an active one, utilizing the available work from the chemical reaction.

Response to Comment on “Boosted molecular mobility during common chemical reactions”

“…We concluded that mobility during several common chemical reactions is more rapid than Brownian diffusion when the free energy release rate exceeds a threshold. This is in pleasing agreement with a master curve that reveals similar boosted mobility of catalytic enzymes, reconciling previous conflicting data in the literature (2). Boosted mobility was confirmed using an independent microfluidic gradient method.…”

Direct Single Molecule Imaging of Enhanced Enzyme Diffusion

“…Recently, this capability of chemical/kinetic energy conversion was also extended to many other types of active enzymes, such as urease, catalase, DNA polymerase, and hexokinase. These enzymes were shown to diffuse faster when catalyzing their substrates in the solution [4][5][6][7][8][9][10][11][12]. The mechanism of the molecular level enhanced diffusion of single enzyme is still under debate.…”

A thermodynamic perspective on enhanced enzyme diffusion

“…Indeed, it reports a strong positive correlation between enhanced diffusion and the change in Gibbs free energy (as well as the Michaelis-Menten reaction rate) from FCS also supported by dynamic light scattering experiments. Such a correlation, whose robustness is further tested by tuning the reaction rate with both temperature and pH, may explain why the effect has been observed for enzymes catalyzing both endothermic as well as exothermic reactions and provides a path toward a satisfactory mechanistic framework, grounded in thermodynamics, of the origin of enhanced diffusion (4). It also unequivocally predicts where future enzymes are expected to fall on the master curve based on the intrinsic properties of the reactions they catalyze.…”

“…Equally surprising is the observation, counter to any macroscopic hydrodynamic expectation, that catalytic proteins (enzymes) exhibit enhanced diffusion upon catalysis (4). The latter is the focus of "Master curve of boosted diffusion for 10 catalytic enzymes" by Jee et al (4), where the effect of diffusion enhancement of 10 enzymes is probed by fluorescence correlation spectroscopy (FCS) (Fig. 1).…”

“…Faster correlation function decays coincide with faster diffusion coefficients. It was this setup that has allowed Jee et al (4) to deduce the diffusion coefficient of 10 different enzymes.…”