Initial mechanistic characterization of Escherichia coli Lon protease

Lon is an ATP-dependent protease which functions in vivo to degrade regulatory proteins, and misfolded or incorrectly translated proteins via endoproteolytic cleavage. Lon is a member of the AAA+ (ATPase Associated Activity) family of proteins. Lon differs from most members of the family in that both the ATPase and the proteolytic domains reside on the same polypeptide chain. Despite a plethora of studies on Lon, no thorough kinetic analysis has been completed. Perhaps the obstacle was the lack of a substrate that would provide quantitative data relevant to the in vivo action of the enzyme. To that end, we synthesized a fluorogenic substrate mimic of lambdaN protein, a known in vivo substrate of Lon protease. This peptide substrate, known as lambdaN89-98FR, is composed of residues 89--98 of the lambdaN sequence and contains a single Lon cleavage site. The hydrolysis of the peptide substrate was directly monitored by an increase in fluorescence corresponding to the separation of the fluorescence resonance energy transfer (FRET) pair that has been incorporated into the peptide. Using lambdaN89-98FR we determined the kinetic parameters kcat and Km for peptide hydrolysis in the presence of saturating ATP. Product inhibition studies were completed using ADP (the product of ATP hydrolysis) and lambdaN94-98, which has the same sequence as the peptide product in the direction of the carboxy terminus. Product inhibition experiments establish what enzyme form(s) (enzyme and/or the enzyme:substrate complex) bind product, and give quantitative binding data for those enzyme forms. Also, we determined the rate of nucleotide hydrolysis using ATP, CTP, UTP, and GTP. Correlating peptidase with NTPase data indicates that Lon undergoes a significant conformational change that allows the energy from nucleotide hydrolysis to be coupled with peptidase activity. This conformational change is dependent on the presence of the optimal nucleotide, ATP. The presence of the conformational change was confirmed by limited trypsin digestion of Lon protease. In addition, isotope exchange experiments were completed to confirm that nucleotide hydrolysis is irreversible. Finally, pre-steady-state peptidase experiments determined the position of peptide hydrolysis relative to the rate-limiting step of the reaction mechanism. The data acquired allow us to propose a complex kinetic mechanism for ATP-dependent proteolysis by Lon protease.