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.
2013
Processive protein degradation is a common feature found in ATP-dependent proteases. This study utilized a physiological substrate of Escherichia coli Lon protease known as the lambda N protein (λN) to initiate the first kinetic analysis of the proteolytic mechanism of this enzyme. To this end, experiments were designed to determine the timing of three selected scissile sites in λN approaching the proteolytic site of ELon and their subsequent cleavages to gain insight into the mechanism by which ATP-dependent proteases attain processivity in protein degradation. The kinetic profile of peptide bond cleavage at different regions of λN was first detected by the iTRAQ/mass spectrometry technique. Fluorogenic λN constructs were then generated as reporter substrates for transient kinetic characterization of the ATP- versus AMPPNP-dependent peptide bond cleavage and the delivery of the scissile sites near the amino- versus carboxyl-terminal of the λN protein to the proteolytic site of ELon. Collectively, our res...
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