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Phosphorylated intermediate of alkaline phosphatase

M Cocivera, J McManaman, I B Wilson

    Biochemistry
    |June 24, 1980
    PubMed
    Summary

    Alkaline phosphatase phosphorylation varies with pH, decreasing from 80% at pH 7 to 9% at pH 10. Key steps influencing reaction rates are chemical dephosphorylation and phosphate dissociation.

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    Area of Science:

    • Biochemistry
    • Enzymology
    • Protein phosphorylation

    Background:

    • Alkaline phosphatase (AP) is a crucial enzyme involved in various biological processes.
    • Understanding the factors affecting AP activity, such as pH and substrate, is essential for its study.

    Purpose of the Study:

    • To investigate the steady-state phosphorylation of alkaline phosphatase subunits.
    • To determine the influence of pH and substrates on AP phosphorylation and reaction kinetics.
    • To elucidate the rate-limiting steps in the AP catalytic mechanism.

    Main Methods:

    • Measurement of alkaline phosphatase subunit phosphorylation under steady-state conditions.
    • Enzyme kinetic analysis using various substrates and pH values.
    • Determination of catalytic rate constants (kcat) to evaluate individual reaction steps.

    Main Results:

    • Phosphorylation levels of AP subunits significantly decreased from 80% at pH 7 to 9% at pH 10.
    • No evidence of anticooperativity was observed between the subunits.
    • Chemical dephosphorylation and phosphate dissociation were identified as the primary rate-influencing steps.
    • The contribution of these steps varied with pH, with phosphate dissociation being significant at neutral pH.

    Conclusions:

    • The study provides quantitative insights into the pH-dependent phosphorylation of alkaline phosphatase.
    • Kinetic analysis reveals that dephosphorylation and phosphate dissociation are key determinants of AP reaction rates.
    • The findings align with recent nuclear magnetic resonance (NMR) studies on phosphate dissociation rates.

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