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Related Experiment Videos

The "phosphoryl-enzyme" from phosphoglycerate kinase

P E Johnson, S J Abbott, J R Knowles

    Biochemistry
    |June 29, 1976
    PubMed
    Summary

    Phosphoglycerate kinase catalysis likely proceeds via a sequential mechanism, not a phosphoryl-enzyme intermediate. Careful interpretation is needed due to potential artifactual results from trace cosubstrates.

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    Area of Science:

    • Biochemistry
    • Enzyme kinetics

    Background:

    • Phosphoglycerate kinase (PGK) is crucial in glycolysis.
    • The mechanism of PGK, particularly the role of a phosphoryl-enzyme intermediate, remains debated.

    Purpose of the Study:

    • To investigate the reaction mechanism of phosphoglycerate kinase.
    • To determine if a stable phosphoryl-enzyme intermediate is formed during catalysis.

    Main Methods:

    • Preparation of a putative "phosphoryl-enzyme" complex using PGK and adenosine 5 omino-triphosphate.
    • Analysis of partial exchange reactions (adenosine 5 omino-diphosphate/adenosine 5 omino-triphosphate and 3-phosphoglycerate/1,3-bisphosphoglycerate).
    • Isoelectric focusing of purified enzyme to correlate catalytic activities.

    Main Results:

    • The "phosphoryl-enzyme" contained stoichiometric 3-phosphoglycerate and was likely a tight complex.
    • Partial exchange reactions were observed but were significantly slower than overall catalysis.
    • Catalysis of exchange reactions and kinase activity coincided post-isoelectric focusing, but required minimal cosubstrate, suggesting artifactual origins.

    Conclusions:

    • Evidence does not support a ping-pong pathway involving a phosphoryl-enzyme intermediate for PGK.
    • A sequential mechanism with phosphoryl transfer within a ternary complex is more likely.
    • Caution is advised when interpreting kinetic data influenced by trace cosubstrates to avoid erroneous conclusions about reaction intermediates.

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