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

Distinct and long-lived activity states of single enzyme molecules

(a) Individual β-galactosidase molecules enclosed in the fiber bundle array exhibited a distinct catalytic activity which was broadly distributed ("static heterogeneity") (see movie (4 MB)). (b) Normalized single molecule substrate turnover distribution of hundreds of single enzymes molecules at different substrate concentrations (vi/‹v› velocity of an individual enzyme molecule / the average velocity; f(v)/c frequency / total count of events). The frequency distributions at substrate concentrations of 25, 50, and 150 µM have the same coefficients of variation (30 ± 1%). Thus, the velocity distribution can be assumed to be a universal function of [S], and - as vi is directly proportional to kcat but not to KM - the single-molecule Michaelis-Menten equation

indicates that variability in kcat but not KM is the source for the wide distribution of vi.

(JACS 2008)

© 2014, Thomas Hirsch, Gisela Emmert.