Monitoring the timing of ATP hydrolysis with activation of peptide cleavage in Escherichia coli Lon by transient kinetics.
Escherichia coli Lon, also known as protease La, is an oligomeric ATP-dependent protease, which functions to degrade damaged and certain short-lived regulatory proteins in the cell. To investigate the kinetic mechanism of E. coli Lon protease, we performed the first pre-steady-state kinetic characterization of the ATPase and peptidase activities of this enzyme. Using rapid quench-flow and fluorescence stopped-flow spectroscopy techniques, we demonstrated that ATP hydrolysis occurs before peptide cleavage, with the former reaction displaying a burst and the latter displaying a lag in product production. The detection of burst kinetics in ATP hydrolysis is indicative of a step after nucleotide hydrolysis being rate-limiting in ATPase turnover. At saturating substrate concentrations, the lag rate constant for peptide cleavage is comparable to the kcat of ATPase, indicating that two hydrolytic processes are coordinated during the first enzyme turnover. The involvement of subunit interaction during enzyme catalysis was detected as positive cooperativity in the binding and hydrolysis of substrates, as well as apparent asymmetry in the ATPase activity in Lon. When our data are taken together, they are consistent with a reaction model in which ATP hydrolysis is used to generate an active enzyme form that hydrolyzes peptide.
Vineyard
Diana
D
Department of Chemistry and Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Patterson-Ward
Jessica
J
Berdis
Anthony J
AJ
Lee
Irene
I
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D004789:Enzyme Activation; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism*; D049070:Protease La / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity; D013997:Time Factors
D029968:Escherichia coli Proteins; D009842:Oligopeptides; D000255:Adenosine Triphosphate; D049070:Protease La; D000251:Adenosine Triphosphatases