Substrate Catalysis Enhances Single-Enzyme Diffusion

Abstract

We show that diffusion of single urease enzyme molecules increases in the presence of urea in a concentration-dependent manner and calculate the force responsible for this increase. Urease diffusion measured using fluorescence correlation spectroscopy increased by 16-28% over buffer controls at urea concentrations ranging from 0.001 to 1 M. This increase was significantly attenuated when urease was inhibited with pyrocatechol, demonstrating that the increase in diffusion was the result of enzyme catalysis of urea. Local molecular pH changes as measured using the pH-dependent fluorescence lifetime of SNARF-1 conjugated to urease were not sufficient to explain the increase in diffusion. Thus, a force generated by self-electrophoresis remains the most plausible explanation. This force, evaluated using Brownian dynamics simulations, was 12 pN per reaction turnover. These measurements demonstrate force generation by a single enzyme molecule and lay the foundation for a further understanding of biological force generation and the development of enzyme-driven nanomotors.

Quantifying the Heat Dissipation from Molecular Motor’s Transport Properties in Nonequilibrium Steady States

“…Compared with freely diffusing exothermic enzymes, kinesin-1 whose dynamics is confined on one-dimensional tracks is highly efficient in transforming conformational fluctuations into a locally directed motion, thus displaying a significantly higher enhancement in diffusivity with its turnover rate. Putting molecular motors and freely diffusing enzymes on an equal footing, our study offers thermodynamic basis to understand the heat enhanced self-diffusion of exothermic enzymes.Together with recent studies [1][2][3][4], Riedel et al [5] have demonstrated a rather surprising result, from a perspective of equilibrium statistical mechanics: Diffusion constants (D) of exothermic enzymes, measured from fluorescence correlation spectroscopy (FCS), increase linearly with their catalytic turnover rates V cat , so that the enhancement of diffusivity at maximal activity (maximum V cat ) is as large aswhere D 0 and D max are the diffusion constants measured by FCS at V cat = 0 and maximal V cat , respectively. Their enigmatic observation [5] has called much attention of biophysics community to the physical origin of the activity-dependent diffusivity of a single enzyme.…”

“…Together with recent studies [1][2][3][4], Riedel et al [5] have demonstrated a rather surprising result, from a perspective of equilibrium statistical mechanics: Diffusion constants (D) of exothermic enzymes, measured from fluorescence correlation spectroscopy (FCS), increase linearly with their catalytic turnover rates V cat , so that the enhancement of diffusivity at maximal activity (maximum V cat ) is as large as…”

Quantitative Analysis of Purine Nucleotides Indicates That Purinosomes Increase de Novo Purine Biosynthesis

“…Two possibilities remain to be explored. The first is reversible posttranslational modification of one or more member of these enzymes; the second is substrate concentration-dependent increase in diffusion of the enzymes in the presence of their respective substrates to drive complex formation (Brownian ratchet) (35)(36)(37). Both are being studied.…”

Hydrodynamic collective effects of active protein machines in solution and lipid bilayers

“…Sen and coworkers (39,40) have shown that catalytically active enzymes have larger diffusion coefficients than their inactive counterparts in the absence of substrate. Recently, chemotaxislike drift of enzymes in the presence of substrate gradients has been observed and used for sorting of the enzymes (41).…”