A tetrahedral transition state at the active sites of the 20S proteasome is coupled to opening of the alpha-ring channel.
Intrinsic conformational transitions contribute to the catalytic action of many enzymes. Here we use a single-molecule approach to demonstrate how such transitions are linked to the catalytic sites of the eukaryotic proteasome, an essential protease of the ubiquitin pathway. The active sites of the cylindrical proteasomal core particle are located in a central chamber accessible through gated entry channels. By using atomic force microscopy, we found continual alternation between open and closed gate conformations. We analyzed the relative abundance of these conformers in wild-type and mutated yeast core particles upon exposure to substrates or inhibitors. Our data indicate that the dynamic gate can be opened by allosteric coupling to a tetrahedral transition state at any of the working active centers. The results point to the N(alpha)-amine of the N-terminal active site threonyl residue as the major effector group responsible for triggering the essential conformational switch.
Osmulski
Pawel A
PA
Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78245, USA.
Hochstrasser
Mark
M
Gaczynska
Maria
M
D000107:Acetylation; D001897:Boronic Acids / Q000494:pharmacology; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D008024:Ligands; D018625:Microscopy, Atomic Force / Q000379:methods*; D015394:Molecular Structure; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D001897:Boronic Acids; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008024:Ligands; C423917:MG 262; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde
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