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Catalytic residues and substrate specificity of recombinant human tripeptidyl peptidase I (CLN2)

Hiroshi Oyama1, Tomoko Fujisawa, Takao Suzuki

  • 1Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku.

Journal of Biochemistry
|August 11, 2005
PubMed

Insights

Tripeptidyl peptidase I (TPP-I), or CLN2, is vital for lysosomal protein breakdown. Its unique substrate-binding cleft and catalytic residues were identified, offering insights into neurodegenerative disease mechanisms.

Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • Tripeptidyl peptidase I (TPP-I), also known as CLN2, is a lysosomal enzyme crucial for protein degradation.
  • Deficiency in TPP-I causes fatal neurodegenerative conditions, highlighting its importance in cellular health.

Purpose of the Study:

  • To elucidate the biochemical role and activity mechanism of TPP-I.
  • To identify key catalytic residues and characterize substrate specificity.

Main Methods:

  • Recombinant human TPP-I and mutant proteins were analyzed.
  • Enzyme kinetics, mutational analyses, and inhibition studies were employed.
  • Sequence similarities with related enzymes were compared.

Main Results:

  • Ser280, Glu77, and Asp81 were identified as essential catalytic residues.
  • TPP-I demonstrated optimal hydrolysis of Ala-Arg-Phe*Nph-Arg-Leu (k(cat)/K(m) = 2.94 microM(-1).s(-1)).
  • The substrate-binding cleft comprises six subsites (S(3)-S(3)'), with a preference for bulky, hydrophobic residues at P(1) and specific residues at P(2).

Conclusions:

  • TPP-I possesses unique substrate-binding properties, including necessary hydrophilic interactions at the S(2) subsite.
  • These findings contribute to understanding TPP-I's function in lysosomal degradation and its potential role in neurodegenerative diseases.

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