title: 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.
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 endo- peptidase 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 S4 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 Ca(2+) 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.
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, 04044-20 São Paulo, Brazil.
authors.forename: Marcia Y
authors.initials: MY
authors.lastname: Kondo
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, 04044-20 São Paulo, Brazil.
authors.forename: Iuri E
authors.initials: IE
authors.lastname: Gouvea
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, 04044-20 São Paulo, Brazil.
authors.forename: Débora N
authors.initials: DN
authors.lastname: Okamoto
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, 04044-20 São Paulo, Brazil.
authors.forename: Jorge A N
authors.initials: JA
authors.lastname: Santos
authors.affiliation: Department of Pharmacology, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, 04044-20 São Paulo, Brazil.
authors.forename: Caden
authors.initials: C
authors.lastname: Souccar
authors.affiliation: Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Oda
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, 04044-20 São Paulo, Brazil.
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, 04044-20 São Paulo, Brazil. Electronic address: map.juliano@gmail.com.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry*; D000818:Animals; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007700:Kinetics; D008297:Male; D059748:Proteolysis; D051381:Rats; D057057:Serine Proteases / Q000737:chemistry*; D014020:Tissue Extracts / Q000737:chemistry; D000091346:Tripeptidyl-Peptidase 1
chemical_list: D014020:Tissue Extracts; C000717269:Tpp1 protein, rat; D000091346:Tripeptidyl-Peptidase 1; D057057:Serine Proteases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C000717267:TPP1 protein, human
keywords: Endopeptidase; FRET peptides; TPP-I; Tripeptidyl amino peptidase