Proceedings Of The National Academy Of Sciences | September 7, 2007

Hans H. Gorris, David M. Rissin and David R. Walt
Proceedings of the National Academy of Sciences
DOI: 10.1021/ja058425e

Abstract: Inhibition kinetics of single-β-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 β-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.