Author |
Kondo, Marcia Y.
![]() ![]() Gouvea, Iuri E. ![]() ![]() Okamoto, Debora N. ![]() ![]() Santos, Jorge A. N. ![]() ![]() Souccar, Caden ![]() ![]() Oda, Kohei ![]() Juliano, Luiz ![]() ![]() Juliano, Maria A. ![]() ![]() |
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 |
|
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 |
|
Access rights | Closed access |
Type | Article |
Web of Science ID | WOS:000369597900010 |
URI | https://repositorio.unifesp.br/handle/11600/58632 |
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