title: Characterization of endopeptidase activity of tripeptidyl peptidase-I/CLN2 protein which is deficient in classical late infantile neuronal ceroid lipofuscinosis.
abstract: Endopeptidase activities of the CLN2 gene product (Cln2p)/tripeptidyl peptidase I (TPP-I), purified from rat spleen, were studied using the synthetic fluorogenic substrates. We designed and constructed decapeptides, based on the known sequence cleavage specificities of bacterial pepstatin-insensitive carboxyl proteases (BPICP). MOCAc-Gly-Lys-Pro-Ile-Pro-Phe-Phe-Arg-Leu-Lys(Dnp)r-NH(2) is readily hydrolyzed by Cln2p/TPP-I (K(cat)/K(m) = 7.8 s(-1) mM(-1)). The enzyme had a maximal activity at pH 3.0 for an endopeptidase substrate, but at pH 4.5 with respect to tripeptidyl peptidase activity. Both endopeptidase and tripeptidyl peptidase activities were strongly inhibited by Ala-Ala-Phe-CH(2)Cl, but not inhibited by tyrostatin, an inhibitor of bacterial pepstatin-insensitive carboxyl proteases, pepstatin, or inhibitors of serine proteases. Fibroblasts from classical late infantile neuronal ceroid lipofuscinosis patients have less than 5% of the normal tripeptidyl peptidase activity and pepstatin-insensitive endopeptidase activity. Cln2p/TPP-I is a unique enzyme with both tripeptidyl peptidase and endopeptidase activities for certain substrate specificity.
authors.affiliation: Department of Biochemistry, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Ezaki
authors.forename: M
authors.initials: M
authors.lastname: Takeda-Ezaki
authors.forename: K
authors.initials: K
authors.lastname: Oda
authors.forename: E
authors.initials: E
authors.lastname: Kominami
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases; D000818:Animals; D002460:Cell Line; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D010450:Endopeptidases / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007223:Infant; D007700:Kinetics; D009472:Neuronal Ceroid-Lipofuscinoses / Q000201:enzymology* / Q000235:genetics; D009842:Oligopeptides / Q000737:chemistry; D010447:Peptide Hydrolases / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D057057:Serine Proteases; D013379:Substrate Specificity; D000091346:Tripeptidyl-Peptidase 1
chemical_list: D009842:Oligopeptides; C000717269:Tpp1 protein, rat; D000091346:Tripeptidyl-Peptidase 1; D010450:Endopeptidases; D010447:Peptide Hydrolases; D057057:Serine Proteases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C000717267:TPP1 protein, human