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Computational Prediction of Amino Acid Preferences of Potentially Multispecific Peptide-Binding Domains Involved in Protein-Protein Interactions
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Functional tyrosine residue in the active center of human dipeptidyl peptidase III

Branka Salopek-Sondi1, Bojana Vukelić, Jasminka Spoljarić

  • 1Division of Molecular Biology, Ruder Bosković Institute, Bijenicka cesta 54, HR-10002 Zagreb, Croatia.

Biological Chemistry
|January 1, 2008
PubMed

Insights

Human dipeptidyl peptidase III (DPP III), a metallopeptidase, has a functional Tyr318 in its active site. This residue is crucial for catalytic activity, suggesting a role in transition state stabilization for M49 peptidases.

Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • Human dipeptidyl peptidase III (DPP III) is a metallopeptidase from family M49.
  • DPP III is implicated in pain modulation and cellular defense against oxidative stress.

Purpose of the Study:

  • To investigate the functional role of Tyr318 in the active site of human DPP III.
  • To elucidate the catalytic mechanism of M49 peptidases.

Main Methods:

  • Heterologous expression of human DPP III.
  • Site-directed mutagenesis of Tyr318 to Phenylalanine (Phe).
  • Enzyme kinetics assays to determine kinetic parameters (kcat, Km) and inhibitor binding affinity.

Main Results:

  • Substitution of Tyr318 with Phe significantly reduced the catalytic rate (kcat) by two orders of magnitude.
  • The mutation did not affect substrate binding affinity (Km).
  • Binding affinity of a competitive hydroxamate inhibitor remained unchanged, indicating Tyr318 is not essential for inhibitor binding to S1/S2 subsites.

Conclusions:

  • Tyr318 plays a critical functional role in the catalytic mechanism of human DPP III.
  • The conserved tyrosine residue is likely involved in stabilizing the transition state during enzymatic catalysis.
  • These findings provide insights into the catalytic mechanism of metallopeptidase family M49.

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