Abstract
Escherichia coli ClpAP protease is an ATP-dependent protease composed of the proteolytic component ClpP and a regulatory ATPase, ClpA. ClpAP protease degraded a variety of peptide bonds in protein and peptide substrates at a slow rate (kcat < or = 30 min-1/subunit of ClpP), but showed very high activity (kcat > or = 800 min-1) for a synthetic peptide composed of the first 19 amino acids of ClpP, MSYSGERDNFAPHMALVPV, referred to as the propeptide. The propeptide was not degraded by ClpP alone, but was degraded in the presence of ClpA and ClpP. Degradation was activated by nonhydrolyzable analogs of ATP, indicating that nucleotide-promoted interaction between ClpA and ClpP is sufficient to activate ClpP for propeptide cleavage. The propeptide, as well as truncated forms lacking either the first 9 or the last 3 amino acids, was cleaved at the same Met-Ala bond at which autoprocessing occurs in vivo. No hydrolysis of FAPHMALVPV derivatives was observed when Met was replaced by Glu, Lys, Ser, Tyr, Ile, and D-Met, but cleavage at the same position did occur with Leu or Trp substitutions. A peptide composed of a tandem repeat of FAPHMALVPV was cleaved between both Met-Ala bonds (Kcat values > or = 39 min-1). Propeptides inhibited degradation of alpha-casein by competition for a binding site on ClpA, and they stimulated the basal ATPase activity of ClpA in the absence of ClpP. Peptides and protein substrates interact at an allosteric site on ClpA, which appears to be the site at which specific substrates are recognized by the Clp protease.
Highlights
D-Met, but cleavage at the same positiondid occur with tein that appears to be related t o subunits of the eukaryotic
Propeptides in- strates t o ClpP or other proteasesor carry out chaperonefunchibited degradation of a-casein by competition for a tions unrelated to degradation[19]
We show that a specific peptide substrate of ClpAP interacts at an allosteric site on ClpA where it competes with protein substrates and that nucleotide-promoted interaction between ClpA and ClpP is sufficient to activate ClpP to degrade the peptide at rates faster than those previously reported for substrates of other ATP-dependent proteases
Summary
The reaction mixture containepdl,acing the coumarin moiety of the fluorogenic substrate with in a final volume of 250 pl, 50 mM Tris-HC1, pH 8.0 (at 25 "C), 25 mM MgCl,, 4 mM ATP, 10 pg of [met/~yl-~Hlcase(i-n70,000 cpm), 0.3 pgof ClpP, and 3.85 pg of ClpA. (1-2 pCi/pmol), and 0.5 pgof ClpA in a total volume of 50 pl Reaction hibited both fluorogenic peptide and a-casein degradation mixtures were incubated for min at 37 "C and thenwere stopped by adding 200 pl of 10%ammonium molybdate in 1M sulfuric acid, 5 m silicotungstic acid. Assay of ClpP PeptidaseActivity-Peptidase activity was measured as described by Woo et al [21]in 50-1.11reaction mixtures containing 1 mM N-succinyl-Leu-Tyr 7-amido-4-methylcoumarin, mM Tris-HC1, pH 8.0 (at 25 "C), 25 mM MgCl,, and 0.5 pg of ClpP.
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