Analysis of catalytic properties of tripeptidyl peptidase I (TTP-I), a serine carboxyl lysosomal protease, and its detection in tissue extracts using selective FRET peptide substrate

Analysis of catalytic properties of tripeptidyl peptidase I (TTP-I), a serine carboxyl lysosomal protease, and its detection in tissue extracts using selective FRET peptide substrate

Author Kondo, Marcia Y. Autor UNIFESP Google Scholar
Gouvea, Iuri E. Autor UNIFESP Google Scholar
Okamoto, Debora N. Autor UNIFESP Google Scholar
Santos, Jorge A. N. Autor UNIFESP Google Scholar
Souccar, Caden Autor UNIFESP Google Scholar
Oda, Kohei Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Juliano, Maria A. Autor UNIFESP Google Scholar
Abstract Tripeptidyl peptidase I (TPP-I), also named ceroid lipofuscinosis 2 protease (CLN2p), is a serine carboxyl lysosomal protease involved in neurodegenerative diseases, and has both tripeptidyl amino- and endopeptidase activities under different pH conditions. We developed fluorescence resonance energy transfer (FRET) peptides using tryptophan (W) as the fluorophore to study TPP-I hydrolytic properties based on previous detailed substrate specificity study (Tian Y. et al., J. Biol. Chem. 2006, 281:6559-72). Tripeptidyl amino peptidase activity is enhanced by the presence of amino acids in the prime side and the peptide NH2-RWFFIQ-EDDnp is so far the best substrate described for TPP-I. The hydrolytic parameters of this peptide and its analogues indicated that the S-4 subsite of TPP-I is occluded and there is an electrostatic interaction of the positively charged substrate N-terminus amino group and a negative locus in the region of the enzyme active site. KCl activated TPP-I in contrast to the inhibition by Ca2+ and NaCl. Solvent kinetic isotope effects (SKIEs) show the importance of the free N-terminus amino group of the substrates, whose absence results in a more complex solvent-dependent enzyme: substrate interaction and catalytic process. Like pure TPP-I, rat spleen and kidney homogenates cleaved NH2-RWFFIQ-EDDnp only at F-F bond and is not inhibited by pepstatin, E-64, EDTA or PMSF. The selectivity of NH2-RWFFIQ-EDDnp to TPP-I was also demonstrated by the 400 times higher k(cat)/K-m compared to generally used substrate, NH2-AAF-MCA and by its resistance to hydrolysis by cathepsin D that is present in high levels in kidneys. (C) 2016 Elsevier Inc. All rights reserved.
Keywords TPP-I
Tripeptidyl amino peptidase
Endopeptidase
FRET peptides
xmlui.dri2xhtml.METS-1.0.item-coverage New York
Language English
Sponsor Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP-Projects)
Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq-Projects)
Grant number FAPESP: 12/50191-4R
FAPESP: 2013/12106-8
CNPq: 471340/2011-1
CNPq: 470388/2010-2
Date 2016
Published in Peptides. New York, v. 76, p. 80-86, 2016.
ISSN 0196-9781 (Sherpa/Romeo, impact factor)
Publisher Elsevier Science Inc
Extent 80-86
Origin https://www.sciencedirect.com/science/article/pii/S0196978116300092
Access rights Closed access
Type Article
Web of Science ID WOS:000369597900010
URI https://repositorio.unifesp.br/handle/11600/58632

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