Kinetic studies of unmodified individual Escherichia coli beta-galactosidase molecules in free solution

Assays were performed on individual Escherichia coli beta-galactosidase molecules at 2 different concentrations of the substrate DDAO-beta-D-galactoside using a free zone capillary electrophoresis-based protocol with an in-laboratory-constructed instrument utilizing laser-induced fluorescence detection. In a typical run, 2 enzyme molecules were injected into the capillary. They were separated from each other by a brief period of electrophoresis and incubated on the capillary in the presence of the substrate. They were then mobilized on the capillary into a zone of substrate at a different concentration, re-incubated, and the product peaks mobilized past the detector . The relative change in activity as the concentration was increased differed between molecules, suggesting differences in Km. In a different experiment, the capillary was filled with on average 13 enzyme molecules per run, incubated, and the activities of the individual molecules determined. The shapes of the distribution curves of single molecule activities obtained at different concentrations of the substrate resorufin-beta-D-galactoside were indistinguishable, suggesting a homogeneous Km. To explain why individual enzyme molecules behaved as if they were heterogeneous with respect to Km but the population behaved as if it were homogeneous, theoretical Michaelis-Menten curves were constructed. The curves for populations with heterogeneous Km values were found to be indistinguishable from that of a homogeneous population.