Substrate Catalysis Enhances Single-Enzyme Diffusion

• Published in: Journal of the American Chemical Society Vol. 132; no. 7; pp. 2110 - 2111
• Main Authors: Muddana, Hari S, Sengupta, Samudra, Mallouk, Thomas E, Sen, Ayusman, Butler, Peter J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 24.02.2010
• Subjects: Benzopyrans - chemistry; Catalysis; Catechols - chemistry; Catechols - metabolism; Catechols - pharmacology; Chemotaxis; Diffusion; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Naphthols - chemistry; Rhodamines - chemistry; Spectrometry, Fluorescence - methods; Urea - chemistry; Urease - antagonists & inhibitors; Urease - chemistry; Urease - metabolism
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja908773a

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.