Application of the sequential n-step kinetic mechanism to polypeptide translocases

Clp/Hsp100 proteins are essential motor proteins in protein quality control pathways in all organisms. Such enzymes couple the energy derived from ATP binding and hydrolysis to translocate and unfold polypeptide substrates. Often they perform this role in collaboration with proteases for protein removal or with other chaperones for protein disaggregation. Unlike other well-characterized motor proteins, fundamental parameters such as the microscopic rate constants and overall rate of translocation, step-size (amino acids translocated per step), processivity, and directionality are not available for many of these enzymes. We have recently developed a fluorescence stopped-flow method to elucidate these fundamental mechanistic details. In addition, we have developed a quantitative method to examine the single-turnover time courses that result from the rapid mixing experiments. With these two advances in hand, we have recently reported the first determination of the microscopic rate constants, overall rate of translocation, kinetic step-size, and processivity for the E. coli ClpA polypeptide translocase. Here, we report a description of both the fluorescence stopped-flow method to examine the mechanism of enzyme catalyzed polypeptide translocation and the mathematics required to quantitatively examine the resulting time courses.