Inter-species variation in the pH dependence of tripeptidyl-peptidase II
(English)Manuscript (preprint) (Other academic)
Tripeptidyl-peptidase II (TPP II) is a large enzyme complex (>4 MDa) participating in the general protein turn-over in the cell downstream of the proteasome. In addition, there have been reports of involvement of TPP II in different physiological situations. To facilitate further investigations of the physiological role of TPP II and its enzymatic properties, a characterization at protein level is necessary. Therefore, an expression system for murine TPP II using Escherichia coli has been developed. The pH-optimum for cleavage of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was investigated for mTPP II, and compared with human TPP II and TPP II from Drosophila melanogaster. It was shown that the mouse enzyme had similar pH dependence as the human enzyme, while dTPP II had a slightly lower optimum. Surprisingly, the investigation also demonstrated that TPP II from all sources showed a different pH-profile for hydrolysis of AAA-pNA compared to AAF-pNA. To investigate this observation further, steady-state kinetic parameters were determined at various pH. Since both the KM and Vmax are lower for cleavage of AAA-pNA, a potential explanation could be that the substrate AAA-pNA is non-productively bound to the active site of the enzyme.
tripeptidyl-peptidase II, TPP II, AAF-pNA, AAA-pNA, steady-state kinetics, pH-dependence
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biochemistry and Molecular Biology
Research subject Biochemistry
IdentifiersURN: urn:nbn:se:uu:diva-134621OAI: oai:DiVA.org:uu-134621DiVA: diva2:373147