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Kinetic characterization of recombinant murine tripeptidyl-peptidase II purified from Pichia pastoris: a comparison with the enzyme from human and Drosophila melanogaster
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry.
Manuscript (Other academic)
Identifiers
URN: urn:nbn:se:uu:diva-95192OAI: oai:DiVA.org:uu-95192DiVA: diva2:169311
Available from: 2006-11-24 Created: 2006-11-24 Last updated: 2010-01-13Bibliographically approved
In thesis
1. Tripeptidyl-Peptidase II: Structure, Function and Gene Regulation
Open this publication in new window or tab >>Tripeptidyl-Peptidase II: Structure, Function and Gene Regulation
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The protein degradation process is of vital importance for the cell to maintain cellular functions. An important enzyme in this process is the multimeric tripeptidyl-peptidase II (TPP II). It removes tripeptides from a free N-terminus of the substrates. TPP II has broad substrate specificity and wide-spread distribution, suggesting that the TPP II gene is a house-keeping gene. However, the levels of both mRNA and TPP II protein varies during different conditions and the TPP II gene promoter was therefore identified and characterized. It is a 215 bp fragment just upstream of the coding sequence. This fragment lacks a TATA-box but contains an initiator, two inverted CCAAT-boxes and an E-box. The CCAAT-boxes and the E-box were found to bind the nuclear factor Y (NF-Y) and upstream stimulatory factor-1 (USF-1) respectively. The CCAAT-boxes appear to be most important for the transcriptional activation. Furthermore, several silencer element were identified further upstream of the 215 bp promoter and the octamer binding factor Oct-1 was found to bind one of these fragments. If Oct-1 is responsible for the inhibition of the transcription of the TPP II gene remains to be investigated. In addition, the substrate specificity was investigated. For this purpose an expression system using Pichia pastoris was developed. The purified recombinant TPP II was found to have the same enzymatic properties as the native enzyme. In order to identify the amino acids involved in the binding of the N-terminus of the substrate, wild-type murine TPP II and four mutants E305Q, E305K, E331Q and E331K were purified. Steady-state kinetic analysis clearly demonstrated that both Glu-305 and Glu-331 are important for this binding as the KMapp is more than 102 higher for the mutants than wild-type. Finally, the pH-dependence for cleavage of two chromogenic substrates was compared for TPP II from different species.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 52 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 243
Keyword
Biochemistry, gene regulation, protein expression, enzyme kinetics, tripeptidyl-peptidase II, proteasome, intracellular protein degradation, exopeptidase, Biokemi
Identifiers
urn:nbn:se:uu:diva-7345 (URN)91-554-6733-4 (ISBN)
Public defence
2006-12-15, B7:113a, BMC, Husargatan 3, Uppsala, 09:15
Opponent
Supervisors
Available from: 2006-11-24 Created: 2006-11-24Bibliographically approved

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