Biochemical characterization of the 20S proteasome from the methanoarchaeon Methanosarcina thermophila.
The 20S proteasome from the methanoarchaeon Methanosarcina thermophila was produced in Escherichia coli and characterized. The biochemical properties revealed novel features of the archaeal 20S proteasome. A fully active 20S proteasome could be assembled in vitro with purified native alpha ring structures and beta prosubunits independently produced in Escherichia coli, which demonstrated that accessory proteins are not essential for processing of the beta prosubunits or assembly of the 20S proteasome. A protein complex with a molecular mass intermediate to those of the alpha7 ring and the 20S proteasome was detected, suggesting that the 20S proteasome is assembled from precursor complexes. The heterologously produced M. thermophila 20S proteasome predominately catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residues Phe and Tyr (chymotrypsinlike activity) in short chromogenic and fluorogenic peptides. Low-level hydrolyzing activities were also detected carboxyl to the acidic residue Asp and the basic residue Arg (trypsinlike activity). Sodium dodecyl sulfate and divalent or monovalent ions stimulated chymotrypsinlike activity and inhibited postglutamyl activity, whereas ATP stimulated postglutamyl activity but had little effect on the chymotrypsinlike activity. The results suggest that the 20S proteasome is a flexible protein which adjusts to binding of substrates. The 20S proteasome also hydrolyzed large proteins. Replacement of the nucleophilic Thr1 residue with an Ala in the beta subunit abolished all activities, which suggests that only one active site is responsible for the multisubstrate activity. Replacement of beta subunit active-site Lys33 with Arg reduced all activities, which further supports the existence of one catalytic site; however, this result also suggests a role for Lys33 in polarization of the Thr1 N, which serves to strip a proton from the active-site Thr1 Ogamma nucleophile. Replacement of Asp51 with Asn had no significant effect on trypsinlike activity, enhanced postglutamyl and trypsinlike activities, and only partially reduced lysozyme-hydrolyzing activity, which suggested that this residue is not essential for multisubstrate activity.
Maupin-Furlow
J A
JA
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802-4500, USA. jmaupin@micro.ifas.ufl.edu
Aldrich
H C
HC
Ferry
J G
JG
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000378:metabolism; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D019641:DNA, Archaeal / Q000032:analysis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D007477:Ions; D008239:Lysine / Q000235:genetics; D017020:Methanosarcina / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D010455:Peptides / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013912:Threonine / Q000235:genetics; D014443:Tyrosine / Q000378:metabolism
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D007477:Ions; D009097:Multienzyme Complexes; D010455:Peptides; D005973:Glutamine; D013912:Threonine; D001224:Aspartic Acid; D012967:Sodium Dodecyl Sulfate; D014443:Tyrosine; D010649:Phenylalanine; D001216:Asparagine; D000255:Adenosine Triphosphate; D001120:Arginine; D003546:Cysteine Endopeptidases; C097046:PsmA protein, Methanosarcina thermophila; C097047:PsmB protein, Methanosarcina thermophila; D008239:Lysine; D000409:Alanine