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 cat alyzes ATP-dependent degradation of proteins. Formation of stable ClpA hexa mers and stable ClpAP complexes requires binding of ATP or nonhydrolyzable ATP analogues to ClpA. To understand the order of events during substrate b inding, unfolding, and degradation by ClpAP, it is essential to know the ol igomeric state of the enzyme during multiple catalytic cycles. Using inacti ve 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 sat urating, the rate constant for dissociation of ClpA hexamers was 0.032 min( -1) (t1/2 of 22 min) at 37 degrees C, and dissociation of ClpP from the Clp AP complexes occurred with a rate constant of 0.092 min(-1) (t1/2 of 7.5 mi n). Because the k(cat) for casein degradation is similar to 10 min(-1), the se 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 dissociat ion, whereas mutations in the C-terminal ATP binding site, which cause sign ificant decreases in ATPase activity, led to lower rates of dissociation of ClpA and ClpAP complexes. Dissociation rates for wild-type and first domai n mutants of ClpA were faster at low nucleotide concentrations. The t1/2 fo r dissociation of ClpAP complexes in the presence of nonhydrolyzable analog ues was greater than or equal to 30 min. Thus, ATP binding stabilizes the o ligomeric state of ClpA, and cycles of ATP hydrolysis affect the dynamics o f oligomer interaction. However, since the k(cat) for ATP hydrolysis is sim ilar to 140 min(-1), ClpA and the ClpAP complex remain associated during hu ndreds of rounds of ATP hydrolysis. Our results indicate that the ClpAP com plex 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.