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1 Department of Biology and
2 Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
Reprint requests to: Robert T. Sauer, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
ClpX requires ATP to unfold protein substrates and translocate them into the proteolytic chamber of ClpP for degradation. The steady-state parameters for hydrolysis of ATP and ATP
S by ClpX were measured with different protein partners and the kinetics of degradation of ssrA-tagged substrates were determined with both nucleotides. ClpX hydrolyzed ATPS to ADP and thiophosphate at a rate (6/min) significantly slower than ATP hydrolysis (140/min), but the hydrolysis of both nucleotides was increased by ssrA-tagged substrates and decreased by ClpP. KM and kcat for hydrolysis of ATP and ATPS were linearly correlated over a 200-fold range, suggesting that protein partners largely affect kcat rather than nucleotide binding, indicating that most bound ATP leaves the enzyme by hydrolysis rather than dissociation, and placing an upper limit of 
15 µM on KD for both nucleotides. Competition studies with ClpX and fluorescently labeled ADP gave inhibition constants for ATPS (2 µM) and ADP (3 µM) under the reaction conditions used for steady-state kinetics. In the absence of Mg2+, where hydrolysis does not occur, the inhibition constant for ATP (55 µM) was weaker but very similar to the value for ATPS (45 µM). Compared with ATP, ATPS supported slow but roughly comparable rates of ClpXP degradation for two Arc-ssrA substrates and denatured GFP-ssrA, but not of native GFP-ssrA. These results show that the processing of protein substrates by ClpX is closely coupled to the maximum rate of nucleotide hydrolysis.
Keywords: AAA+ ATPase; catalyzed protein unfolding; ATP-dependent proteolysis; motor proteins; mantADP
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