title: Distinct and long-lived activity states of single enzyme molecules.
abstract: Individual enzyme molecules have been observed to possess discrete and different turnover rates due to the presence of long-lived activity states. These stable activity states are thought to result from different molecular conformations or post-translational modifications. The distributions in kinetic activity observed in previous studies were obtained from small numbers of single enzyme molecules. Due to this limitation, it has not been possible to fully characterize the different kinetic and equilibrium binding parameters of single enzyme molecules. In this paper, we analyze hundreds of single beta-galactosidase molecules simultaneously; using a high-density array of 50,000 fL-reaction chambers, we confirm the presence of long-lived kinetic states within a population of enzyme molecules. Our analysis has isolated the source of kinetic variability to kcat. The results explain the kinetic variability within enzyme molecule populations and offer a deeper understanding of the unique properties of single enzyme molecules. Gaining a more fundamental understanding of how individual enzyme molecules work within a population should provide insight into how they affect downstream biochemical processes. If the results reported here can be generalized to other enzymes, then the stochastic nature of individual enzyme molecule kinetics should have a substantial impact on the overall metabolic activity within a cell.
authors.affiliation: Department of Chemistry, Tufts University, Medford, Massachusetts 02155, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Rissin
authors.forename: Hans H
authors.initials: HH
authors.lastname: Gorris
authors.forename: David R
authors.initials: DR
authors.lastname: Walt
mesh_terms: D002384:Catalysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D012996:Solutions; D001616:beta-Galactosidase / Q000378:metabolism*
chemical_list: D012996:Solutions; D001616:beta-Galactosidase