ClpA and ClpP remain associated during multiple rounds of ATP-dependent protein degradation by ClpAP protease.

The Escherichia coli ClpA and ClpP proteins form a complex, ClpAP, that catalyzes ATP-dependent degradation of proteins. Formation of stable ClpA hexamers and stable ClpAP complexes requires binding of ATP or nonhydrolyzable ATP analogues to ClpA. To understand the order of events during substrate binding, unfolding, and degradation by ClpAP, it is essential to know the oligomeric state of the enzyme during multiple catalytic cycles. Using inactive forms of ClpA or ClpP as traps for dissociated species, we measured the rates of dissociation of ClpA hexamers or ClpAP complexes. When ATP was saturating, the rate constant for dissociation of ClpA hexamers was 0.032 min(-1) (t(1/2) of 22 min) at 37 degrees C, and dissociation of ClpP from the ClpAP complexes occurred with a rate constant of 0. 092 min(-1) (t(1/2) of 7.5 min). Because the k(cat) for casein degradation is approximately 10 min(-1), these results indicate that tens of molecules of casein can be turned over by the ClpAP complex before significant dissociation occurs. Mutations in the N-terminal ATP binding site led to faster rates of ClpA and ClpAP dissociation, whereas mutations in the C-terminal ATP binding site, which cause significant decreases in ATPase activity, led to lower rates of dissociation of ClpA and ClpAP complexes. Dissociation rates for wild-type and first domain mutants of ClpA were faster at low nucleotide concentrations. The t(1/2) for dissociation of ClpAP complexes in the presence of nonhydrolyzable analogues was >/=30 min. Thus, ATP binding stabilizes the oligomeric state of ClpA, and cycles of ATP hydrolysis affect the dynamics of oligomer interaction. However, since the k(cat) for ATP hydrolysis is approximately 140 min(-1), ClpA and the ClpAP complex remain associated during hundreds of rounds of ATP hydrolysis. Our results indicate that the ClpAP complex is the functional form of the protease and as such engages in multiple rounds of interaction with substrate proteins, degradation, and release of peptide products without dissociation.