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Measuring In Vitro ATPase Activity for Enzymatic Characterization
07:38

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Published on: August 23, 2016

Enzymatic activation of autotaxin by divalent cations without EF-hand loop region involvement

J Lee1, I D Jung, S W Nam

  • 1College of Medicine, Konyang University, Nonsan 320-711, South Korea.

Biochemical Pharmacology
|June 8, 2001
PubMed

Insights

Autotaxin (ATX), an enzyme stimulating tumor cell motility, requires divalent cations like Ca(2+) or Mg(2+) for optimal activity. These cations enhance enzyme kinetics, with effects likely mediated outside the EF-hand loop region.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Autotaxin (ATX) is a nucleotide pyrophosphatase/phosphodiesterase (NPP) enzyme.
  • ATX exhibits potent tumor cell motility-stimulating activity.
  • ATX possesses homology to phosphodiesterase (PDE) catalytic sites and an EF-hand motif.

Purpose of the Study:

  • To investigate the influence of the EF-hand loop region on ATX enzymatic activity.
  • To determine the role of divalent cations in modulating ATX enzymatic functions.
  • To elucidate the mechanism by which cations affect ATX activity.

Main Methods:

  • Enzyme kinetics assays were performed on wild-type and mutant ATX proteins.
  • The effects of calcium (Ca2+) and magnesium (Mg2+) ions were assessed.
  • Chelating agents were used to evaluate the requirement for divalent cations.
  • Site-directed mutagenesis and deletion mutations were introduced in the EF-hand loop region.

Main Results:

  • Divalent cations (Ca2+, Mg2+) significantly increased ATX PDE activity in a concentration-dependent manner.
  • Chelation of divalent cations abolished ATX enzymatic activity.
  • Mutations within the EF-hand loop region did not significantly alter enzymatic activity.
  • A deletion mutant of the EF-hand loop retained cation sensitivity, suggesting cation interaction sites are outside this region.

Conclusions:

  • Divalent cations are essential for optimal ATX enzymatic activity.
  • The EF-hand loop region is not the primary site for cation-mediated enhancement of ATX activity.
  • Cation-dependent regulation of ATX activity likely involves interactions with other parts of the protein.

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