© 2005 The Japanese Biochemical Society
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Catalytic Residues and Substrate Specificity of Recombinant Human Tripeptidyl Peptidase I (CLN2)
1 Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585; 2 Chuo-Sanken Laboratory, Katakura Industries Co., Ltd. Sayama, Saitama 350-1352; 3 Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, Florida 32610-0245, USA; and 4 Protein Structure Section, Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, Maryland 21702-1201, USA
* To whom correspondence should be addressed. Tel: +81-75-724-7763, Fax: +81-75-724-7760, E-mail: bika{at}ipc.kit.ac.jp
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 µM–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 (S3-S3'). TPP-I prefers bulky and hydrophobic amino acid residues at the P1 position and Ala, Arg, or Asp at the P2 position. Hydrophilic interactions at the S2 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.
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