Peptidylglutamyl-peptide hydrolase activity of the multicatalytic proteinase complex: evidence for a new high-affinity site, analysis of cooperative kinetics, and the effect of manganese ions.
The multicatalytic proteinase (MCP) complex or proteasome is a major nonlysosomal proteinase of eukaryotic cells. The proteinase can cleave peptide bonds on the carboxyl side of hydrophobic, basic, or acidic amino acid residues. These activities have been referred to as "chymotrypsin-like", "trypsin-like", and "peptidylglutamyl-peptide hydrolase" activities, respectively, and have been shown to be catalyzed at distinct sites. The latter activity is often assayed with the synthetic peptide substrate Z-Leu-Leu-Glu-beta-naphthylamide (LLE-NA). N-tBoc-Ala-Ala-Asp-SBzl is also a substrate for the rat liver MCP, suggesting a broader specificity for cleavage on the carboxyl side of acidic residues than the peptidylglutamyl-peptide hydrolase activity previously reported. The pH optimum is in the range of pH 7.0-7.5. Studies of the dependence of velocity on LLE-NA concentration show (a) that there is a high-affinity site (LLE1) which obeys Michaelis-Menten kinetics with a Km value of approximately 100 microM and (b) that at higher substrate concentrations (LLE2) the curve is sigmoidal, suggesting either allosteric activation of the proteinase at a second site or the involvement of multiple catalytic sites which display positive cooperativity. Activity at the high-affinity site (LLE1) can be distinguished from that of the activity of the LLE2 component by the effect of inhibitors, divalent metal ions, and KCl, as well as by its response to heat treatment. The addition of 1 mM MnCl2 stimulates both LLE1 and LLE2 activities and also permits saturation of MCP with substrate at concentrations of LLE-NA below the solubility limit of this peptide.(ABSTRACT TRUNCATED AT 250 WORDS)
Djaballah
H
H
Department of Biochemistry, University of Leicester, U.K.
Rivett
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002413:Cations, Divalent / Q000494:pharmacology; D003374:Coumarins / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D049934:Isocoumarins; D008099:Liver / Q000201:enzymology; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D011189:Potassium Chloride; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D011919:Rats, Inbred Strains
D002413:Cations, Divalent; D003374:Coumarins; D049934:Isocoumarins; D008670:Metals; D009097:Multienzyme Complexes; D009842:Oligopeptides; D011480:Protease Inhibitors; C037284:benzyloxycarbonylleucyl-leucyl-glutamine-2-naphthylamide; C046235:3,4-dichloroisocoumarin; D011189:Potassium Chloride; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex