University of Regensburg Trenner Faculty of Chemistry Trenner Analytical Chemistry
Single molecule analysis in femtoliter arrays

Stochastic inhibitor release and binding from single-enzyme molecules

In a pre-steady-state experiment a bulk solution of β-galactosidase was preincubated with 100 µM of the slow binding inhibitor D-galactal ( ≈ 5 × Ki). Enzyme and inhibitor were diluted 1000-fold with substrate solution and enclosed in the fiber bundle array. Sequential fluorescence images of single enzyme substrate turnovers were taken every 30 s (see movie (500 kB)). (A) A sequence of these images shows a delayed onset of substrate turnover that can be attributed to stochastic inhibitor release events. (B) Fluorescence increase in the indicated femtoliter-containers (red: container without enzyme). (C) Off-time distribution. (D) The semi-logarithmic plot illustrates a first-order inhibitor release.

A steady-state experiment with c(D-galactal) ≈ Ki, revealed repeated inhibitor binding and release events (see movie (1 MB)), which are accompanied by conformational changes of the enzyme’s catalytic site. We proved that the rate constants of inhibitor release and binding derived from stochastic changes in the substrate turnover are consistent with bulk reaction kinetics.

(PNAS 2007)

© 2014, Thomas Hirsch, Gisela Emmert.