Identification of a region in the N-terminus of Escherichia coli Lon that affects ATPase, substrate translocation and proteolytic activity.

Lon, also known as protease La, is an AAA+ protease machine that contains the ATPase and proteolytic domain within each enzyme subunit. Three truncated Escherichia coli Lon (ELon) mutants were generated based on a previous limited tryptic digestion result and hydrogen-deuterium exchange mass spectrometry analyses performed in this study. Using methods developed for characterizing wild-type (WT) Lon, we compared the ATPase, ATP-dependent protein degradation and ATP-dependent peptidase activities. With the exception of not degrading a putative structured substrate known as CcrM (cell-cycle-regulated DNA methyltransferase), the mutant lacking the first 239 residues behaved like WT ELon. Comparing the activity data of WT and ELon mutants reveals that the first 239 residues are not needed for minimal enzyme catalysis. The mutants lacking the first 252 residues or residues 232-252 displayed compromised ATPase, protein degradation and ATP-dependent peptide translocation abilities but retained WT-like steady-state peptidase activity. The binding affinities of WT and Lon mutants were evaluated by determining the concentration of λ N (K(λN)) needed to achieve 50% maximal ATPase stimulation. Comparing the K(λN) values reveals that the region encompassing 232-252 of ELon could contribute to λ N binding, but the effect is modest. Taken together, results generated from this study reveal that the region constituting residues 240-252 of ELon is important for ATPase activity, substrate translocation and protein degradation.

Cheng I ，Mikita N ，Fishovitz J ，Frase H ，Wintrode P ，Lee I ... -  《-》

Functional role of the N-terminal region of the Lon protease from Mycobacterium smegmatis.

Roudiak SG ，Shrader TE 《BIOCHEMISTRY》

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

Thomas J ，Fishovitz J ，Lee I 《-》

Processive degradation of unstructured protein by Escherichia coli Lon occurs via the slow, sequential delivery of multiple scissile sites followed by rapid and synchronized peptide bond cleavage events.

Mikita N ，Cheng I ，Fishovitz J ，Huang J ，Lee I ... -  《-》

Transient kinetic experiments demonstrate the existence of a unique catalytic enzyme form in the peptide-stimulated ATPase mechanism of Escherichia coli Lon protease.

Vineyard D ，Zhang X ，Lee I 《BIOCHEMISTRY》