A comparative study of the chymotrypsin-like activity of the rat liver multicatalytic proteinase and the ClpP from Escherichia coli.
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.
Arribas
J
J
Departamento de Bioquímica, Facultad de Medicina de la Universidad Autonoma de Madrid, Spain.
Castaño
J G
JG
D049069:ATP-Dependent Proteases; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification; D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000378:metabolism*; D003429:Cross Reactions; D003546:Cysteine Endopeptidases / Q000276:immunology / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006360:Heat-Shock Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000276:immunology / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D012697:Serine Endopeptidases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D013379:Substrate Specificity
D000906:Antibodies; D006360:Heat-Shock Proteins; D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex