Lack of proteasome active site allostery as revealed by subunit-specific inhibitors.
The chymotrypsin-like (CT-L) activity of the proteasome is downregulated by substrates of the peptidyl-glutamyl peptide hydrolyzing (PGPH) activity. To investigate the nature of such interactions, we synthesized selective alpha',beta'-epoxyketone inhibitors of the PGPH activity. In cellular proliferation and protein degradation assays, these inhibitors revealed that selective PGPH inhibition was insufficient to inhibit protein degradation, indicating that the CT-L and PGPH sites function independently. We also demonstrated that CT-L inhibition by a PGPH substrate does not require the occupancy of the PGPH site or hydrolysis of the PGPH substrate. Thus, these results support a model in which a substrate of one subunit regulates the activity of another via binding to a noncatalytic site(s) rather than through binding to an active site.
Myung
J
J
Department of Molecular, Cellular, and Developmental Biology, New Haven, CT 06520, USA.
Kim
K B
KB
Lindsten
K
K
Dantuma
N P
NP
Crews
C M
CM
D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism; D004852:Epoxy Compounds / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D007659:Ketones / Q000494:pharmacology; D007700:Kinetics; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013379:Substrate Specificity; D014162:Transfection
D004852:Epoxy Compounds; D007659:Ketones; D009097:Multienzyme Complexes; D011480:Protease Inhibitors; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C116212:dihydroeponemycin; D012694:Serine; D010450:Endopeptidases; D002918:Chymotrypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C114177:peptidylglutamylpeptide hydrolase