Coupling of proteolysis to ATP hydrolysis upon Escherichia coli Lon protease functioning. I. Kinetic aspects of ATP hydrolysis

Some aspects of the ATPase function of the Escherichia coli Lon protease we re studied around the optimum pH value. It was revealed that, in the absenc e of the protein substrate, the maximum ATPase activity of the enzyme is ob served at an equimolar ratio of ATP and Mg2+ ions in the area of their mill imolar concentrations. Free components of the substrate complex (ATP-Mg)(2- ) inhibit the enzyme ATPase activity. It is hypothesized that the effector activity of free Mg2+ ions is caused by the formation of the "ADP-Mg-form" of the ATPase centers. It was shown that the activation of ATP hydrolysis i n the presence of the protein substrate is accompanied by an increase in th e affinity of the (ATP-Mg)(2-) complex to the enzyme, by the elimination of the inhibiting action of free Mg2+ ions without altering the efficiency of catalysis of ATP hydrolysis (based on the k(cat) value), and by st change in the type of inhibition of ATP hydrolysis by the (ADP-Mg)(-) complex (wit hout changing the K-i value). Interaction of the Lon protease protein subst rate with the enzyme area located outside the peptide hydrolase center was demonstrated by a direct experiment.