Energy-dependent degradation: Linkage between ClpX-catalyzed nucleotide hydrolysis and protein-substrate processing

ClpX requires ATP to unfold protein substrates and translocate them into the proteolytic chamber of CIpP for degradation. The steady-state parameters for hydrolysis of ATP and ATP-gammaS by ClpX were measured with different protein partners and the kinetics of degradation of ssrA-tagged substrates were determined. with both nucleotides. ClpX hydrolyzed ATPgammaS 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. K-M and k(cat) for hydrolysis of ATP and ATPgammaS were linearly correlated over a 200-fold range, suggesting that protein partners largely affect k(cat) rather than nucleotide binding, indicating that most bound ATP leaves the enzyme by hydrolysis rather than dissociation, and placing an upper limit of approximate to15 muM on K-D for both nucleotides. Competition studies with ClpX and fluorescently labeled ADP gave inhibition constants for ATPgammaS (=2 muM) and ADP (approximate to3 muM) under the reaction conditions used for steady-state kinetics. In the absence of Mg2+, where hydrolysis does not occur, the inhibition constant for ATP (approximate to55 muM) was weaker but very similar to the value for ATRgammaS (approximate to45 muM). Compared with ATP, ATPgammaS 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.