Utilization of positional isotope exchange experiments to evaluate reversibility of ATP hydrolysis catalyzed by Escherichia coli Lon protease

Abstract

Lon protease, also known as protease La, is an ATP-dependent serine protease. Despite the presence of a proteolytic Ser-Lys dyad, the enzyme only catalyzes protein degradation in the presence of ATP. Lon possesses an intrinsic ATPase activity that is stimulated by protein and certain peptide substrates. Through sequence alignment and analysis, it is concluded that Lon belongs to the AAA+ protein family. Previous kinetic characterization of the ATPase domain of Escherichia coli Lon protease implicates a half-site reactivity model in which only 50% of the ATP bound to Lon are hydrolyzed to yield ADP; the remaining ATPase sites remain bound with ATP and are considered non-catalytic. In this model, it is implied that ATP hydrolysis is irreversible. To further evaluate the proposed half-site reactivity model, the reversibility of the ATPase activity of E. coli Lon was evaluated by positional isotope exchange experiments. The ATPase reactions were conducted in the 18O-enriched buffer such that the extent of 18O incorporation into inorganic phosphate generated from ATP hydrolysis could be used to evaluate the extent of reversibility in ATP hydrolysis. Collectively, our experimental data reveal that the ATPase reaction catalyzed by E. coli Lon in the presence and absence of peptide substrate that stimulated the enzyme's ATPase activity is irreversible. Therefore, the half-site ATPase reactivity of E. coli Lon is validated, and can be used to account for the kinetic mechanism of the ATP-dependent peptidase activity of the enzyme.