An Examination of the Kinetic Mechanism of the ATP-Dependent Protease ClpAP

Abstract

Motor proteins perform functions within the cell by converting the energy of ATP into mechanical work. One such example of this can be found in the ATP-dependent protease from Escherichia coli, ClpAP, which is assembled from two distinct enzymes, a protein unfoldase, ClpA, and a protease, ClpP. The biologically active complex functions through a coordinated action in which ClpA is responsible for enzyme catalyzed protein unfolding and ATP-dependent polypeptide translocation, while ClpP will proteolytically degrade polypeptide substrates that have been translocated into its central cavity. Without such systems, deleterious effects are often observed within the cell as a result of either the accumulation of misfolded proteins or from unregulated activity from partially synthesized proteins.Of interest is the kinetic mechanism of ClpAP, and more specifically, the possibility of allosteric effects of ClpP upon ClpA. Kinetic parameters such as the step-size of polypeptide translocation, processvity, macroscopic rate, and microscopic rate constants have been measured for ClpA in the absence of ClpP under single-turnover conditions, but quantitative estimates of these parameters have not been determined in the presence of the proteolytic component, ClpP. In an effort to address this issue, single-turnover chemical quenched-flow methods in conjunction with stopped-flow fluorescence techniques have been used to examine the molecular mechanism of ClpAP catalyzed polypeptide translocation and degradation. The single-turnover methods are performed with enzyme in large excess over substrates and allow for the observation of the conversion of substrates into intermediates or products in a single cycle of catalysis. We have shown that the kinetic parameters are dramatically different in the presence of the proteolytic component and ClpP has a clear allosteric effect on the mechanism.