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J Bacteriol, March 1998, p. 1480-1487, Vol. 180, No. 6
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Biochemical Characterization of the 20S Proteasome from the Methanoarchaeon Methanosarcina thermophila

Julie A. Maupin-Furlow,^1,2,* Henry C. Aldrich,² and James G. Ferry^1,

Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802-4500,¹ and Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611-0700²

Received 3 November 1997/Accepted 16 January 1998

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  ring structures and  prosubunits independently produced in Escherichia coli, which demonstrated that accessory proteins are not essential for processing of the  prosubunits or assembly of the 20S proteasome. A protein complex with a molecular mass intermediate to those of the ₇ 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  subunit abolished all activities, which suggests that only one active site is responsible for the multisubstrate activity. Replacement of  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 O 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.

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^* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802-4500. Phone: (352) 392-4095. Fax: (352) 392-5922. E-mail: jmaupin{at}micro.ifas.ufl.edu.

Corresponding author. Mailing address: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611-0700. Phone: (814) 863-5721. Fax: (814) 863-6217. E-mail: jgf3{at}psu.edu.

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