Binding of hydrophobic peptides to several non-catalytic sites promotes peptide hydrolysis by all active sites of 20 S proteasomes: Evidence for peptide-induced channel opening in the alpha-rings
Binding of hydrophobic peptides to several non-catalytic sites promotes peptide hydrolysis by all active sites of 20 S proteasomes: Evidence for peptide-induced channel opening in the alpha-rings
The eukaryotic 20 S proteasome contains the following 6 active sites: 2 chymotrypsin-like, 2 trypsin-like, and 2 caspase-like. We previously showed that hydrophobic peptide substrates of the chymotrypsin-like sites allosterically stimulate peptide hydrolysis by the caspase-like sites and their own cleavage. More thorough analysis revealed that these peptides also stimulate peptide hydrolysis by the trypsin-like site. This general activation by hydrophobic peptides occurred even if the chymotrypsin-like sites were occupied by a covalent inhibitor and was highly cooperative, with an average Hill coefficient of 7. Therefore, this stimulation of peptide hydrolysis at all active sites occurs upon binding of hydrophobic peptides to several non-catalytic sites. The stimulation by hydrophobic peptides was not observed in the yeast Delta N alpha 3 mutant 20 S proteasomes, in 20 S-PA26 complexes, or SDS-activated proteasomes and was significantly lower in 26 S proteasomes, all of which appear to have the gated channel in the alpha-rings in an open conformation and hydrolyze peptides at much faster rates than 20 S proteasomes. Also the hydrophobic peptides altered K(m), V(max) of active sites in a similar fashion as PA26 and the Delta N alpha 3 mutation. The activation by hydrophobic peptides was decreased in K(+)-containing buffer, which favors the closed state of the channels. Therefore, hydrophobic peptides stimulate peptide hydrolysis most likely by promoting the opening of the channels in the alpha-rings. During protein breakdown, this peptide-induced channel opening may function to facilitate the release of products from the proteasome.
Allosteric Site, Animals, Binding Sites, Caspases/chemistry, Catalytic Domain, Chymotrypsin/chemistry, Cysteine Endopeptidases/chemistry, Dose-Response Relationship, Drug, Hydrolysis, Kinetics, Multienzyme Complexes/chemistry, Muscles/metabolism, Mutation, Peptide Hydrolases/chemistry, Peptides/chemistry, Potassium Chloride/pharmacology, Proteasome Endopeptidase Complex, Protein Binding, Protein Conformation, Rabbits, Recombinant Proteins/chemistry, Saccharomyces cerevisiae/metabolism, Time Factors, Trypsin/chemistry
22260-22270
Kisselev, Alexei F.
5c90995e-b51a-4aec-b3c5-2b4c3a5e2266
Kaganovich, Daniel
ebb13f4e-e925-4aef-88e7-ddc25ef52d8f
Goldberg, Alfred L.
d96ae983-fc7f-43f1-a3ec-a7f8c3530304
21 June 2002
Kisselev, Alexei F.
5c90995e-b51a-4aec-b3c5-2b4c3a5e2266
Kaganovich, Daniel
ebb13f4e-e925-4aef-88e7-ddc25ef52d8f
Goldberg, Alfred L.
d96ae983-fc7f-43f1-a3ec-a7f8c3530304
Kisselev, Alexei F., Kaganovich, Daniel and Goldberg, Alfred L.
(2002)
Binding of hydrophobic peptides to several non-catalytic sites promotes peptide hydrolysis by all active sites of 20 S proteasomes: Evidence for peptide-induced channel opening in the alpha-rings.
The Journal of Biological Chemistry, 277 (25), .
(doi:10.1074/jbc.M112360200).
Abstract
The eukaryotic 20 S proteasome contains the following 6 active sites: 2 chymotrypsin-like, 2 trypsin-like, and 2 caspase-like. We previously showed that hydrophobic peptide substrates of the chymotrypsin-like sites allosterically stimulate peptide hydrolysis by the caspase-like sites and their own cleavage. More thorough analysis revealed that these peptides also stimulate peptide hydrolysis by the trypsin-like site. This general activation by hydrophobic peptides occurred even if the chymotrypsin-like sites were occupied by a covalent inhibitor and was highly cooperative, with an average Hill coefficient of 7. Therefore, this stimulation of peptide hydrolysis at all active sites occurs upon binding of hydrophobic peptides to several non-catalytic sites. The stimulation by hydrophobic peptides was not observed in the yeast Delta N alpha 3 mutant 20 S proteasomes, in 20 S-PA26 complexes, or SDS-activated proteasomes and was significantly lower in 26 S proteasomes, all of which appear to have the gated channel in the alpha-rings in an open conformation and hydrolyze peptides at much faster rates than 20 S proteasomes. Also the hydrophobic peptides altered K(m), V(max) of active sites in a similar fashion as PA26 and the Delta N alpha 3 mutation. The activation by hydrophobic peptides was decreased in K(+)-containing buffer, which favors the closed state of the channels. Therefore, hydrophobic peptides stimulate peptide hydrolysis most likely by promoting the opening of the channels in the alpha-rings. During protein breakdown, this peptide-induced channel opening may function to facilitate the release of products from the proteasome.
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Published date: 21 June 2002
Keywords:
Allosteric Site, Animals, Binding Sites, Caspases/chemistry, Catalytic Domain, Chymotrypsin/chemistry, Cysteine Endopeptidases/chemistry, Dose-Response Relationship, Drug, Hydrolysis, Kinetics, Multienzyme Complexes/chemistry, Muscles/metabolism, Mutation, Peptide Hydrolases/chemistry, Peptides/chemistry, Potassium Chloride/pharmacology, Proteasome Endopeptidase Complex, Protein Binding, Protein Conformation, Rabbits, Recombinant Proteins/chemistry, Saccharomyces cerevisiae/metabolism, Time Factors, Trypsin/chemistry
Identifiers
Local EPrints ID: 474266
URI: http://eprints.soton.ac.uk/id/eprint/474266
ISSN: 0021-9258
PURE UUID: 3776007c-9d53-41e5-866f-1e75470a988f
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Date deposited: 16 Feb 2023 18:08
Last modified: 17 Mar 2024 04:17
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Author:
Alexei F. Kisselev
Author:
Daniel Kaganovich
Author:
Alfred L. Goldberg
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