Kinetic and Molecular Properties of Lysine-sensitive Aspartokinase

Abstract

Abstract The sedimentation coefficient of the lysine-sensitive aspartokinase of Escherichia coli B in sucrose gradients containing lysine increases with increasing temperature and ionic strength. Sedimentation equilibrium molecular weight determinations provide the basis for interpreting the sedimentation velocity results as being due to a rapidly reversible, lysine-mediated association reaction predominantly of the monomer-dimer type. The dependencies of dimerization and lysine binding on lysine concentration are almost identical, with the half-maximal response occurring around 30 µm. Hill coefficients for lysine inhibition demonstrate a temperature dependence which parallels that for dimerization. Maximum dimerization and maximum Hill coefficients are obtained at temperatures above 20°. Arrhenius plots of enzyme activity are biphasic with a break occurring at 20°. The response of the kinetics of lysine inhibition to the parameters influencing enzyme structure together with the stoichiometry of lysine binding (2 lysines per protein dimer or per 4 subunits) suggest that the association reaction is related to the cooperativity of lysine inhibition.