Kinetic analysis of substrate competition in enzymatic reactions with β-D-galactosidase by capillary electrophoresis / dynamic frontal analysis

Abstract

Competitive inhibition between two substrates with an enzyme is investigated by capillary electrophoresis/dynamic frontal analysis (CE/DFA). Enzymatic hydrolyses of o-nitrophenyl β-D-galactopyranoside and p-nitrophenyl β-D-galactopyranoside with β-D-galactosidase were examined as a model competitive reaction. A sample solution containing the two substrates was injected into a capillary filled with a separation buffer containing an enzyme. Enzymatic hydrolysis occurred during the electrophoresis, and the products of o-nitrophenol and p-nitrophenol were continuously formed and resolved from the sample zone. Two-steps plateau signal was detected with the two-substrate solutions based on the difference in the effective electrophoretic mobility of o-nitrophenol and p-nitrophenol. Michaelis-Menten constants and inhibition constants were determined with the plateau heights. Usefulness of CE/DFA on competitive inhibition analysis is demonstrated in this study.