title: Stochastic inhibitor release and binding from single-enzyme molecules.
abstract: Inhibition kinetics of single-beta-galactosidase molecules with the slow-binding inhibitor d-galactal have been characterized by segregating individual enzyme molecules in an array of 50,000 ultra small reaction containers and observing substrate turnover changes with fluorescence microscopy. Inhibited and active states of beta-galactosidase could be clearly distinguished, and the large array size provided very good statistics. With a pre-steady-state experiment, we demonstrated the stochastic character of inhibitor release, which obeys first-order kinetics. Under steady-state conditions, the quantitative detection of substrate turnover changes over long time periods revealed repeated inhibitor binding and release events, 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.
authors.affiliation: Department of Chemistry, Tufts University, 62 Talbot Avenue, Medford, MA 02155, USA.
authors.forename: Hans H
authors.initials: HH
authors.lastname: Gorris
authors.forename: David M
authors.initials: DM
authors.lastname: Rissin
authors.forename: David R
authors.initials: DR
authors.lastname: Walt
mesh_terms: D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism*; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism*; D005697:Galactosides / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D013269:Stochastic Processes; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D004798:Enzymes; D005697:Galactosides; D001616:beta-Galactosidase