Single molecule analysis in femtoliter arrays

**(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 (v_{i}/‹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 v_{i} is directly proportional to *k*_{cat} but not to *K*_{M} - the single-molecule Michaelis-Menten equation

indicates that variability in *k*_{cat} but not *K*_{M} is the source for the wide distribution of v_{i}.

© 2014, Thomas Hirsch, Gisela Emmert.