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Progress curve analysis in enzyme kinetics: model discrimination and parameter estimation

R G Duggleby, J F Morrison

    Biochimica Et Biophysica Acta
    |October 12, 1978
    PubMed
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    This study extends progress curve analysis for enzyme kinetics to reversible two-substrate reactions. The method accurately determined kinetic parameters and the mechanism for aspartate aminotransferase.

    Area of Science:

    • Biochemistry
    • Enzyme kinetics
    • Biophysical chemistry

    Background:

    • The Duggleby and Morrison method (1977) is a standard for analyzing enzyme-catalyzed reaction progress curves.
    • Extending this method to reversible, two-substrate reactions is crucial for understanding complex enzymatic processes.
    • Aspartate aminotransferase (EC 2.6.1.1) is a key enzyme involved in amino acid metabolism.

    Purpose of the Study:

    • To adapt and validate the progress curve analysis method for a reversible, two-substrate enzyme reaction.
    • To determine the kinetic parameters and mechanism of aspartate aminotransferase using this extended method.
    • To improve the accuracy of standard error calculations in progress curve analysis.

    Main Methods:

    • Enzyme-catalyzed recycling of alpha-ketoglutarate to glutamate using glutamate dehydrogenase, NADH, and NH4Cl.

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  • Application of the extended progress curve analysis to the aspartate aminotransferase reaction.
  • Development of a data compression procedure to obtain more realistic standard errors for kinetic parameters.
  • Fitting compressed data to integrated rate equations to determine the kinetic mechanism.

    • Main Results:

    • Kinetic parameters for aspartate aminotransferase determined by progress curve analysis closely matched steady-state velocity measurements.
    • The original method's standard errors underestimated experimental variation; the data compression procedure yielded more realistic estimates.
    • The Ping-Pong kinetic model provided the best fit for the aspartate aminotransferase reaction data.

    Conclusions:

    • Progress curve analysis, when extended and refined, is a robust method for determining kinetic parameters of enzyme-catalyzed reactions.
    • The study confirms the Ping-Pong mechanism for aspartate aminotransferase.
    • The developed data compression technique enhances the reliability of kinetic parameter estimations and their associated errors.