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Related Experiment Videos

Phosphorylase phosphatase. Comparison of active forms using peptide substrates.

S J McNall1, E H Fischer

  • 1Department of Biochemistry, University of Washington, Seattle 98195.

The Journal of Biological Chemistry
|February 5, 1988
PubMed
Summary

Rabbit skeletal muscle phosphorylase phosphatase activity is modulated by Mn2+ and phosphorylation. The enzyme

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Area of Science:

  • Biochemistry
  • Enzymology
  • Protein Phosphorylation

Background:

  • Phosphorylase phosphatase (PP1) is a key enzyme in glycogen metabolism.
  • PP1 activity is regulated by various mechanisms, including phosphorylation and metal ions.
  • Understanding PP1 substrate specificity is crucial for elucidating its physiological roles.

Purpose of the Study:

  • To investigate the substrate specificity of different activated forms of phosphorylase phosphatase.
  • To explore the role of Mn2+ ions and regulatory subunit phosphorylation in enzyme activation and substrate recognition.

Main Methods:

  • Purification of rabbit skeletal muscle phosphorylase phosphatase.
  • Enzyme activation using trypsin-Mn2+ or factor FA (glycogen synthase kinase 3) with Mg2+-ATP.
  • Assays using protein substrates (phosphorylase a, regulatory subunit of cAMP-dependent protein kinase) and synthetic phosphopeptides.
  • Characterization of enzyme activity with varying Mn2+ concentrations.

Main Results:

  • Trypsin-Mn2+ activated enzyme and Mn2+-activated catalytic subunit dephosphorylated both protein substrates and synthetic phosphopeptides.
  • Factor FA-activated enzyme, considered physiologically relevant, dephosphorylated protein substrates but not phosphopeptides.
  • Mn2+ ions were essential for the catalytic subunit to act on phosphopeptides, with optimal effect at 250 microM.
  • Mg2+ and Ca2+ could not substitute for Mn2+ in peptide dephosphorylation.
  • All enzyme forms readily dephosphorylated p-nitrophenyl phosphate, indicating no difference in specificity for this artificial substrate.

Conclusions:

  • The substrate recognition mechanism of the FA-activated phosphorylase phosphatase is more complex than just the primary sequence of the phosphorylation site.
  • Protein substrates likely possess additional determinants recognized by the FA-activated enzyme.
  • Mn2+ ions may relax conformational constraints in the enzyme, enabling broader substrate accessibility, particularly for smaller phosphopeptides.

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