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Related Experiment Videos

Function assignment to conserved residues in mammalian alkaline phosphatases.

Alexey Kozlenkov1, Thomas Manes, Marc F Hoylaerts

  • 1Department of Medical Biosciences, Umeå University, S-901 85 Umeå, Sweden.

The Journal of Biological Chemistry
|April 9, 2002
PubMed
Summary

Investigating human placental alkaline phosphatase (PLAP) and E. coli alkaline phosphatase (ECAP) reveals key residue functions. Mutations highlight differences in metal ion binding and disulfide bond roles, impacting enzyme activity and stability.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • Human placental alkaline phosphatase (PLAP) and E. coli alkaline phosphatase (ECAP) share structural similarities but exhibit functional differences.
  • Understanding the structure-function relationship of key residues is crucial for enzyme mechanism elucidation.

Purpose of the Study:

  • To probe the structural and functional roles of key residues in PLAP by comparing them with homologous residues in ECAP.
  • To investigate the impact of specific mutations on enzyme activity, stability, and inhibitor binding.

Main Methods:

  • Site-directed mutagenesis of wild-type PLAP and a variant (G429) mimicking human germ cell alkaline phosphatase.
  • Substitution of active site metal ligands and neighboring residues with alanines or homologous ECAP residues.

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  • Analysis of enzyme kinetics, heat stability, and inhibition by uncompetitive inhibitors.

    • Main Results:

    • Mutations at Zn2 or Mg sites affected PLAP and ECAP similarly, but the Zn1 ion environment in PLAP was less sensitive to substitution.
    • Disrupting the Cys-121-Cys-183 disulfide bond abolished enzyme activity, while the Cys-467-Cys-474 bond had a lesser structural role.
    • The Tyr-367 residue, unique to mammalian APs, is critical for subunit contact, heat stability, and uncompetitive inhibitor binding.

    Conclusions:

    • Key residues, including metal ligands and cysteines involved in disulfide bonds, play distinct roles in PLAP structure and function compared to ECAP.
    • The Tyr-367 residue is vital for the stability and inhibitory properties of mammalian alkaline phosphatases.
    • Mutagenesis and computational approaches provide insights into the molecular basis of alkaline phosphatase activity and inhibition.