Related Experiment Video

Updated: Jul 15, 2025

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050
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X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

Published on: May 13, 2020

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Elucidating the Role of a Calcium-Binding Loop in an x-Prolyl Aminodipeptidase from Lb. helveticus

Stephanie Ryder1, Jacob Pedigo1, Deanna Dahlke Ojennus1

  • 1Department of Chemistry, Whitworth University, 300 W. Hawthorne Rd., Spokane, Washington 99251, United States.

ACS Omega
|October 2, 2023
PubMed

Insights

Calcium binding to prolyl aminopeptidase (PepX) is crucial for its enzymatic activity. Mutational analysis revealed that while some calcium-binding residues are non-essential, specific mutations render the enzyme inactive, highlighting calcium

Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • Prolyl aminopeptidase (PepX) is an alpha/beta hydrolase enzyme.
  • PepX cleaves peptide bonds at the penultimate N-terminal prolyl position.
  • A conserved calcium-binding motif (xDxDxDGxxD) exists in PepX from lactic acid bacteria, but its function is unknown.

Purpose of the Study:

  • To investigate the role of the conserved calcium-binding loop in the function of Lactobacillus helveticus PepX.
  • To determine if calcium ions are essential for PepX enzymatic activity and stability.

Main Methods:

  • Site-directed mutagenesis was used to create D196A and D194A/D196A mutants.
  • Enzyme activity was assessed using colorimetric kinetic assays.
  • Protein thermal shift assays were employed to evaluate enzyme stability.

Main Results:

  • The D196A mutation did not significantly affect PepX activity or stability compared to the wild-type enzyme.
  • The D194A/D196A double mutant was completely inactive, despite maintaining native-like structure and thermal stability.
  • These findings contradicted initial observations with EDTA and calcium titrations.

Conclusions:

  • Calcium binding to PepX appears to be essential for its catalytic activity.
  • The D194A/D196A mutation disrupts a critical calcium-binding interaction necessary for enzyme function.
  • Further investigation is needed to reconcile the structural and biochemical data regarding calcium's role in PepX.

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