Catalytic residues and substrate specificity of recombinant human tripeptidyl peptidase I (CLN2).
Tripeptidyl peptidase I (TTP-I), also known as CLN2, a member of the family of serine-carboxyl proteinases (S53), plays a crucial role in lysosomal protein degradation and a deficiency in this enzyme leads to fatal neurodegenerative disease. Recombinant human TPP-I and its mutants were analyzed in order to clarify the biochemical role of TPP-I and its mechanism of activity. Ser280, Glu77, and Asp81 were identified as the catalytic residues based on mutational analyses, inhibition studies, and sequence similarities with other family members. TPP-I hydrolyzed most effectively the peptide Ala-Arg-Phe*Nph-Arg-Leu (*, cleavage site) (k(cat)/K(m) = 2.94 microM(-1).s(-1)). The k(cat)/K(m) value for this substrate was 40 times higher than that for Ala-Ala-Phe-MCA. Coupled with other data, these results strongly suggest that the substrate-binding cleft of TPP-I is composed of only six subsites (S(3)-S(3)'). TPP-I prefers bulky and hydrophobic amino acid residues at the P(1) position and Ala, Arg, or Asp at the P(2) position. Hydrophilic interactions at the S(2) subsite are necessary for TPP-I, and this feature is unique among serine-carboxyl proteinases. TPP-I might have evolved from an ancestral gene in order to cleave, in cooperation with cathepsins, useless proteins in the lysosomal compartment.
Oyama
Hiroshi
H
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku.
Fujisawa
Tomoko
T
Suzuki
Takao
T
Dunn
Ben M
BM
Wlodawer
Alexander
A
Oda
Kohei
K
D000626:Aminopeptidases; D000818:Animals; D012831:Bombyx; D020134:Catalytic Domain; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D010450:Endopeptidases; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011994:Recombinant Proteins; D057057:Serine Proteases; D013379:Substrate Specificity; D000091346:Tripeptidyl-Peptidase 1
D010455:Peptides; D011994:Recombinant Proteins; D000091346:Tripeptidyl-Peptidase 1; D010450:Endopeptidases; D010447:Peptide Hydrolases; D057057:Serine Proteases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C000717267:TPP1 protein, human