| Full text | |
| Author(s): |
Kondo, Marcia Y.
[1]
;
Gouvea, Iuri E.
[1]
;
Okamoto, Debora N.
[1]
;
Santos, Jorge A. N.
[1]
;
Souccar, Caden
[2]
;
Oda, Kohei
[3]
;
Juliano, Luiz
[1]
;
Juliano, Maria A.
[1]
Total Authors: 8
|
| Affiliation: | [1] Univ Fed Sao Paulo, Escola Paulista Med, Dept Biophys, Rua Tres de Maio 100, BR-0404420 Sao Paulo - Brazil
[2] Univ Fed Sao Paulo, Escola Paulista Med, Dept Pharmacol, Rua Tres de Maio 100, BR-0404420 Sao Paulo - Brazil
[3] Kyoto Inst Technol, Dept Appl Biol, Kyoto 606 - Japan
Total Affiliations: 3
|
| Document type: | Journal article |
| Source: | Peptides; v. 76, p. 80-86, FEB 2016. |
| Web of Science Citations: | 0 |
| 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. (AU) | |
| FAPESP's process: | 13/12106-8 - Screening and characterization of serine carboxyl peptidases and glutamic peptidases in bacteria isolated from the composting operation at the São Paulo Zoo Park |
| Grantee: | Márcia Yuri Kondo |
| Support Opportunities: | Scholarships in Brazil - Post-Doctoral |
| FAPESP's process: | 12/50191-4 - Synthesis, kinetic studies and applications of substrates and inhibitors for proteolytic enzymes |
| Grantee: | Maria Aparecida Juliano |
| Support Opportunities: | Research Projects - Thematic Grants |