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  Navigation inside a protease: Substrate selection and product exit in the tricorn protease from Thermoplasma acidophilum

Kim, J. S., Groll, M., Musiol, H.-J., Behrendt, R., Kaiser, M., Moroder, L., et al. (2002). Navigation inside a protease: Substrate selection and product exit in the tricorn protease from Thermoplasma acidophilum. Journal of Molecular Biology, 324(5), 1041-1050.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-6DA9-B Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-6DAA-9
Genre: Journal Article
Alternative Title : J. Mol. Biol.

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 Creators:
Kim, J. S.1, Author              
Groll, M.1, Author              
Musiol, H.-J.2, Author              
Behrendt, R.2, Author              
Kaiser, M.2, Author              
Moroder, L.2, Author              
Huber, R.1, Author              
Brandstetter, H.1, Author              
Affiliations:
1Huber, Robert / Structure Research, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565155              
2Moroder, Luis / Bioorganic Chemistry, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565160              

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Free keywords: crystal structure; enzymatic mechanism; hydrolase; beta- propeller; tricorn protease
 Abstract: The proposed pathway and mechanism of substrate entry and product egress in the hexameric D3 symmetric tricorn protease from Thermoplasma acidophilum were explored by crystallographic studies of ligand complexes and by structure-based mutagenesis. Obstruction of the pore within the 7-bladed beta-propeller (beta7) domain by alkylation or oxidation of an engineered double cysteine mutant strongly decreased enzymatic activities. In line herewith, the crystal structure of the tricorn protease in complex with a trideca-peptide inhibitor modifying the catalytic Ser965 revealed part of the peptide trapped inside the channel of the beta7 domain. The cysteine mutation widening the lumen of the 6-bladed beta-propeller (beta6) domain enhanced catalytic activity, which was restored to normal values after its alkylation. A charge reversal mutant at the putative anchor site of the substrate C terminus, R131E-R132E, drastically reduced the proteolytic activity. The complex crystal structure of a peptide inhibitor with a diketo group at the cleavage site mapped the substrate recognition site and confirmed the role of Arg131-Arg132 as an anchor site. Our results strongly suggest the wider beta7 domain to serve as a selective filter and guide of the substrate to the sequestered active site, while the narrower beta6 domain routes the product to the surface. Moreover, we identified the role of Arg131- Arg132 in anchoring the substrate C terminus. (C) 2002 Elsevier Science Ltd. All rights reserved.

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Language(s): eng - English
 Dates: 2002-12-13
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: eDoc: 34902
ISI: 000179960200012
 Degree: -

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Title: Journal of Molecular Biology
  Alternative Title : J. Mol. Biol.
Source Genre: Journal
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Publ. Info: -
Pages: - Volume / Issue: 324 (5) Sequence Number: - Start / End Page: 1041 - 1050 Identifier: ISSN: 0022-2836