Abstract
A comparative study of the chymotrypsin-like activity of the purified recombinant ClpP protease and the multicatalytic proteinase from rat liver is presented. The peptidase activity of both enzymes has been analyzed with several synthetic fluorogenic peptides, containing either aromatic or nonpolar amino acids in their P1 position. The respective Vmax, Km, and Vmax/Km were calculated from kinetic experiments. The substrate specificity of the multicatalytic proteinase, as expressed by Vmax/Km values, indicate the following substrate preference: N-Suc-IIW-MCA > N-Suc-LY-MCA > N-Suc-LLVY-MCA > or = N-Suc-AAF-MCA > N-Cbz-GGL-beta-NA > Glut-GGF-beta-NA > FPAM-4-MNA. In the case of the ClpP the order of preference is: N-Suc-LY-MCA > N-Suc-IIW-MCA > N-Suc-LLVY-MCA > or = N-Suc-AAF-MCA > or = N-Cbz-GGL-beta-NA > FPAM-4-MNA (where: N-Suc, N-succinyl-; MCA, 7-amido-4-methyl coumarin; beta-NA, beta-naphthylamide; N-Cbz, N-benzyloxycarbonyl-; 4-MNA, 4-methoxy-beta-naphthylamide; Glut, glutaryl. This similar substrate specificity is further supported by the lack of activity of both enzymes against SY-MCA and N-Suc-AAPF-MCA (known substrates of chymotrypsin), by very reduced activity against N-Suc-AAA-MCA and by no significant activity against LG-beta-NA. The results of mixed substrate experiments have shown that all the peptides that are substrates seem to be hydrolyzed by a single class of chymotrypsin-like site in both enzymes. The substrate specificity studies suggest a possible evolutionary relationship between the catalytic component of the ClpP of Escherichia coli and the multicatalytic proteinase chymotrypsin-like catalytic component. This conclusion is further supported by other circumstantial evidence: the fact that affinity-purified anti-ClpP antibodies cross-react with two polypeptide components of the rat liver multicatalytic proteinase complex, presented here and also shown previously; the known resemblance of both structures at the electron microscope level; and their reported role in the degradation of NH2-end rule substrates.
Highlights
A comparative studyof the chymotrypsin-like activ- whose carboxyl side contains basic, hydrophobic and acid ity of the purified recombinant ClpP proteaseand the amino acids; the first two specificities are often referred to as multicatalytic proteinase from rat liver is presented. the trypsin-like and chymotrypsin-like
ClpP obtained from the final purification step (DEAE-HPLC, see above) was injected (200 pl) on a gel filtration HPLC column (TSK 5000, Beckman) equilibrated in 50 mM Tris-C1, pH 8.0, 0.3 M KC1, 1 identical to the one reported previously (8).C shows the log plot used to calculate the native molecular mass of the purified recombinant ClpP protein,which is 240 kDa, and D shows an analysis by SDS-PAGE of the indicated fractions from the ChymotrypsinS-lpikeecificity of the MCP and the ClpP
Increasing equation, based on the results presented here and those by the length of the hydrophobic amino acids, but keeping Y in Rivett (22), the expected value for that quotient would be 4. the P1 position, substrate N-Suc-LLVY-MCA produces a The concordance is quitesatisfactory,takinginto account drastic decrease in the K, of ClpP for this substrate (75 p ~ )that we are comparing results from three different laboratoand in theV
Summary
For expression of the recombinant ClpP protein,the ClpP gene was excised from the plasmid pGClpP by digestion with BamHI and subcloned into thepT7-7 expression vector (14),digested with the same restriction enzyme. Affinity-purified anti-ClpP antibodies were obtained as follows: purified recombinant ClpPprotein was subjected to 15% SDS-PAGE, Western-blotted, stained with Ponceau Red, and a horizontal strip containing the ClpP protein excised,blocked in blocking buffer (see above), and incubated with theanti-ClpP antiserum (0.5 ml)in blocking buffer. The reaction mixture contained in a final volume of 0.5 ml: 50 mM Tris-C1,pH 7.4, 1mM DTT, different concentrations of the fluorogenic peptides, andthe appropriate amounts of the purified rat liver MCP or ClpP (2-4pg) obtained from the last step of purification (DEAE-HPLC, see above). Sonication (three times, 30 s each), glycerol wasthen added to a final concentration of 10% (v/v) and centrifuged at 100,000 X g for 60 min
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