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.

Figure 1

(A) Diffusion coefficient of urease increased with increasing substrate concentration. (B) Increase in urease diffusion coefficient was significantly attenuated by the urease inhibitor, pyrocatechol. Error bars represent standard deviation. * indicates a significance value of P < 0.05

Figure 2

Mean square displacement of urease under no reaction (black) and maximum reaction rate assuming a force of 12 pN/turnover (red) obtained from Brownian dynamics simulations. Solid lines are linear fits at longer time scales.

Figure 3

SNARF-1 fluorescence lifetime was measured as a function of solution pH (inset). Catalysis of urea was accompanied by a significant increase in pH (relative to [urea] = 0.001 M). This increase was significantly attenuated by pyrocatechol for all urea concentrations tested. Error bars represent standard deviation.