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Investigation of a role for Glu-331 and Glu-305 in substrate binding of tripeptidyl-peptidase II
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2008 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1784, no 12, 1899-1907 p.Article in journal (Refereed) Published
Abstract [en]

The aim of this study was to investigate the mechanism by which tripeptidyl-peptidase II (TPP II) can specifically release tripeptides from the free N-terminus of an oligopeptide. The subtilisin-like N-terminal part of TPP II was modelled using subtilisin as template. Two glutamate residues (Glu-305 and Glu-331) appeared to be positioned so as to interact with the positively charged N-terminus of the substrate. In order to test this potential interaction, both residues were replaced by glutamine and lysine. The catalytic efficiency was reduced 400-fold for the E331Q variant and 20000-fold for the E331K variant, compared with the wild-type (wt). A substantial part of this reduction was due to decreased substrate affinity, since the K(M) for both mutants was at least two orders of magnitude greater than for the wt. This decrease was linked specifically to interaction with the free N-terminal amino group, based on inhibition studies. Glu-305 appears to be essential for enzymatic activity, but the extremely low activity of the E305Q variant prevented an investigation of the involvement of Glu-305 in substrate binding. The present work is, to our knowledge, the first report to investigate a mechanism for a tripeptidyl-peptidase activity through site-directed mutagenesis.

Place, publisher, year, edition, pages
2008. Vol. 1784, no 12, 1899-1907 p.
Keyword [en]
Tripeptidyl-peptidase II, Exopeptidase, Substrate specificity, Intracellular protein degradation, Oligomerization, Molecular ruler
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-100055DOI: 10.1016/j.bbapap.2008.08.017ISI: 000261673300002PubMedID: 18822395OAI: oai:DiVA.org:uu-100055DiVA: diva2:209321
Available from: 2009-03-24 Created: 2009-03-24 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Interpreting a Giant: Studies of Structure and Function of Tripeptidyl-peptidase II
Open this publication in new window or tab >>Interpreting a Giant: Studies of Structure and Function of Tripeptidyl-peptidase II
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Tripeptidyl-peptidase II (TPP II) is a subtilisin-like serine peptidase that forms a gigantic homooligomeric complex, and is involved in the degradation of peptides in the cytosol. In addition, TPP II has been implicated in specific cellular processes, such as apoptosis control and adipogenesis, but if this is dependent on its endo- or exopeptidase activity remains to be determined. This work is devoted to the structure and function of TPP II, and to finding connections between the two.

Evolutionarily conserved regions of TPP II have been identified, and sequence signatures have been constructed as an aid in identification of TPP II homologues. The conserved regions highlight amino acid residues of potential importance to structure, function or both. In addition, the first TPP II homologue in a prokaryote has been documented, which was likely the result of a horizontal gene transfer.

Substrate binding for the exopeptidase activity of TPP II has been studied through mutagenesis of Glu-331, which revealed a molecular ruler mechanism that positions substrates for cleavage at the third peptide bond from the N-terminus. Thus, the well-known tripeptidyl-releasing property of TPP II could be explained. The exopeptidase activity was also probed by pH dependence studies, which revealed that a substrate with a smaller residue in the P1 position could bind non-productively to the active site. Furthermore, a difference in the pH dependence of KM between TPP II from Drosophila and homologues from mammals indicated a difference in the configuration of the binding pockets between these species.

The endopeptidase activity of TPP II has also been investigated, and was found to differ from the exopeptidase activity. The endopeptidase activity appeared to be promiscuous and the preference for basic amino acid residues in the P1 position reported earlier could not be substantiated.

In conclusion, many structural and mechanistic features have been observed in this work. This might be of value to future drug discovery efforts towards TPP II, and in elucidating the physiological role of this gigantic enzyme.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 45 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 791
Keyword
Tripeptidyl-peptidas II, molecular ruler, sequence signatures, pH-dependence, endopeptidase
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-134633 (URN)978-91-554-7966-4 (ISBN)
Public defence
2011-01-21, B7:101a, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Note
Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 721Available from: 2010-12-22 Created: 2010-11-30 Last updated: 2011-03-21Bibliographically approved

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Tomkinson, Birgitta

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