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 p roperties revealed novel features of the archaeal 20S proteasome, A fu ll active 20S proteasome could be assembled in vitro with purified nat ive alpha ring structures and beta prosubunits independently produced in Escherichia coli, which demonstrated that accessory proteins are no t essential for processing of the beta prosubunits or assembly of the 20S proteasome, A protein complex with a molecular mass intermediate t o those of the alpha(7) ring and the 20S proteasome was detected, sugg esting that the 20S proteasome is assembled from precursor complexes. The heterologously produced M. thermophila 20S proteasome predominatel y catalyzed cleavage of peptide bonds carboxyl to the acidic residue G lu (postglutamyl activity) and the hydrophobic residues Phe and Tyr (c hymotrypsinlike activity) in short chromogenic and fluorogenic peptide s, 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 chym otrypsinlike activity and inhibited postglutamyl activity, whereas ATP stimulated postglutamyl activity but had little effect on the chymotr ypsinlike activity, The results suggest that the 20S proteasome is a f lexible protein which adjusts to binding of substrates. The 20S protea some also hydrolyzed large proteins, Replacement of the nucleophilic T hr(1) residue with an Ala in the beta subunit abolished all activities , which suggests that only one active site is responsible for the mult isubstrate activity. Replacement of beta subunit active-site Lys(33) w ith Arg reduced all activities, which further supports the existence o f one catalytic site; however, this result also suggests a role for Ly s(33) in polarization of the Thr(1) N, which serves to strip a proton from the active-site Thr(1) Oy nucleophile. Replacement of Asp(51) wit h Asn had no significant effect on trypsin like activity, enhanced pos tglutamyl and trypsinlike activities, and only partially reduced lysoz yme-hydrolyzing activity, which suggested that this residue is not ess ential for multisubstrate activity.