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.

2016 
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 NH 2 -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 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 NH 2 -RWFFIQ-EDDnp only at F F bond and is not inhibited by pepstatin, E-64, EDTA or PMSF. The selectivity of NH 2 -RWFFIQ-EDDnp to TPP-I was also demonstrated by the 400 times higher k cat / K M compared to generally used substrate, NH 2 -AAF-MCA and by its resistance to hydrolysis by cathepsin D that is present in high levels in kidneys.
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