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The Active Sites of the Eukaryotic 20 S Proteasome and Their Involvement in Subunit Precursor Processing
- Source :
- Journal of Biological Chemistry. 272:25200-25209
- Publication Year :
- 1997
- Publisher :
- Elsevier BV, 1997.
-
Abstract
- The 26 S proteasome is the central protease involved in ubiquitin-mediated protein degradation and fulfills vital regulatory functions in eukaryotes. The proteolytic core of the complex is the 20 S proteasome, a cylindrical particle with two outer rings each made of 7 different alpha-type subunits and two inner rings made of 7 different beta-type subunits. In the archaebacterial 20 S proteasome ancestor proteolytically active sites reside in the 14 uniform beta-subunits. Their N-terminal threonine residues, released by precursor processing, perform the nucleophilic attack for peptide bond hydrolysis. By directed mutational analysis of 20 S proteasomal beta-type proteins of Saccharomyces cerevisiae, we identified three active site-carrying subunits responsible for different peptidolytic activities as follows: Pre3 for post-glutamyl hydrolyzing, Pup1 for trypsin-like, and Pre2 for chymotrypsin-like activity. Double mutants harboring only trypsin-like or chymotrypsin-like activity were viable. Mutation of two potentially active site threonine residues in the Pre4 subunit excluded its catalytic involvement in any of the three peptidase activities. The generation of different, incompletely processed forms of the Pre4 precursor in active site mutants suggested that maturation of non-active proteasomal beta-type subunits is exerted by active subunits and occurs in the fully assembled particle. This trans-acting proteolytic activity might also account for processing intermediates of the active site mutated Pre2 subunit, which was unable to undergo autocatalytic maturation.
- Subjects :
- Threonine
Proteasome Endopeptidase Complex
Genotype
Macromolecular Substances
medicine.medical_treatment
Protein subunit
DNA Mutational Analysis
Molecular Sequence Data
Restriction Mapping
Saccharomyces cerevisiae
Biology
Protein degradation
Polymerase Chain Reaction
Biochemistry
Multienzyme Complexes
medicine
Animals
Chymotrypsin
Amino Acid Sequence
Binding site
Molecular Biology
Peptide sequence
DNA Primers
Mammals
Enzyme Precursors
Binding Sites
Protease
Sequence Homology, Amino Acid
Active site
Cell Biology
Archaea
Recombinant Proteins
Models, Structural
Cysteine Endopeptidases
Proteasome
Mutagenesis, Site-Directed
biology.protein
Protein Processing, Post-Translational
Sequence Alignment
Gene Deletion
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 272
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....2177335d58f898b5c490b13ea07d151a