Effect of Size, Quaternary Structure and Translational Error on the Static and Dynamic Heterogeneity of β-Galactosidase and Measurement of Electrophoretic Dynamic Heterogeneity

Abstract

Single enzyme molecule assays were performed using capillary electrophoresis-based protocols on beta-galactosidase from Lactobacillus delbrueckii, Lactobacillus reuteri, Lactobacillus helveticus and Bacillus circulans. The enzyme was found to show static heterogeneity with respect to catalytic rate and the variance in rate increased with protein size. This is consistent with the proposal that random errors in translation may be an important underlying component of enzyme heterogeneity. Additionally these enzymes were found to show static heterogeneity with respect to electrophoretic mobility. Comparison of wild-type and rpsL E. coli beta-galactosidase expressed in the presence and absence of streptomycin suggested that increases in error do not result in detectable increases in the dynamic heterogeneity of activity with increasing temperature. Finally, a method was developed to measure the dynamic heterogeneity in electrophoretic mobility.

Arrhenius Plot for a Reaction Catalyzed by a Single Molecule of β-Galactosidase

“…The negative value indicates that the electrophoretic mobility opposes the EOF, and mobility increases in strength with a rise in temperature. In a previous study, quadruple static incubation single molecule β-galactosidase assays were performed at different temperatures. The spacings between the peaks was used to calculate the electrophoretic mobilities of the enzyme molecules at the different temperatures as described previously .…”