WFRF:(Tomkinson Birgitta)
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Exploring the active site of tripeptidyl-peptidase II through studies of pH dependence of reaction kinetics
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- Eklund, Sandra (författare)
- Uppsala universitet,Institutionen för medicinsk biokemi och mikrobiologi,Institutionen för biokemi och organisk kemi
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- Lindås, Ann-Christin (författare)
- Uppsala universitet,Molekylär evolution
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- Hamnevik, Emil (författare)
- Uppsala universitet,Biokemi
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- Elsevier BV, 2012
- 2012
- Engelska.
- Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639 .- 1878-1454. ; 1824:4, s. 561-570
- Tidskriftsartikel (refereegranskat)
Abstract
Ämnesord
Stäng
- Tripeptidyl-peptidase II (TPP II) is a subtilisin-like serine protease which forms a large enzyme complex (> 4 MDa). It is considered a potential drug target due to its involvement in specific physiological processes. However, information is scarce concerning the kinetic characteristics of TPP II and its active site features, which are important for design of efficient inhibitors. To amend this, we probed the active site by determining the pH dependence of TPP II catalysis. Access to pure enzyme is a prerequisite for kinetic investigations and herein we introduce the first efficient purification system for heterologously expressed mammalian TPP II. The pH dependence of kinetic parameters for hydrolysis of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was determined for murine, human and Drosophila melanogaster TPP II as well as mutant variants thereof. The investigation demonstrated that TPP II, in contrast to subtilisin, has a bell-shaped pH dependence of kcatapp/KM probably due to deprotonation of the N-terminal amino group of the substrate at higher pH. Since both the KM and kcatapp are lower for cleavage of AAA-pNA than for AAF-pNA we propose that the former can bind non-productively to the active site of the enzyme, a phenomenon previously observed with some substrates for subtilisin. Two mutant variants, H267A and D387G, showed bell-shaped pH-dependence of kcatapp, possibly due to an impaired protonation of the leaving group. This work reveals previously unknown differences between TPP II orthologues and subtilisin as well as features that might be conserved within the entire family of subtilisin-like serine peptidases.
Ämnesord
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
Nyckelord
- tripeptidyl-peptidase II
- AAF-pNA
- AAA-pNA
- steady-state kinetics
- pH-dependence
- Biokemi
- Biochemistry
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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- Biochimica et Biophysica Acta - Proteins and Proteomics (Sök värdpublikationen i LIBRIS)
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- Eklund, Sandra
- Lindås, Ann-Chri ...
- Hamnevik, Emil
- Widersten, Mikae ...
- Tomkinson, Birgi ...
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