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Related Experiment Videos

Active human-yeast chimeric phosphoglycerate kinases engineered by domain interchange.

M T Mas, C Y Chen, R A Hitzeman

    Science (New York, N.Y.)
    |August 15, 1986
    PubMed
    Summary

    Hybrid enzymes combining human and yeast phosphoglycerate kinase (PGK) domains retain similar catalytic properties. This indicates evolutionary changes do not significantly impact PGK

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    Area of Science:

    • Biochemistry
    • Enzymology
    • Structural Biology

    Background:

    • Phosphoglycerate kinase (PGK) is a crucial enzyme in glycolysis, existing as a monomer with two distinct domains.
    • PGK undergoes a substrate-induced conformational change, closing its active site cleft located between the two domains.
    • Significant amino acid differences exist between human and yeast PGK (35% divergence).

    Purpose of the Study:

    • To investigate the relationship between the structure and function of phosphoglycerate kinase (PGK).
    • To assess the impact of interspecies domain swapping on PGK's catalytic properties and active site.
    • To determine if evolutionary substitutions affect key enzymatic mechanisms like domain closure and activity.

    Main Methods:

    • Construction of two interspecies hybrid PGK enzymes, each with one domain from human PGK and one from yeast PGK.

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  • Comparative analysis of the catalytic properties of the hybrid enzymes against their parental human and yeast PGK counterparts.

    • Main Results:

    • The constructed hybrid PGK enzymes exhibited catalytic properties highly similar to those of the original human and yeast enzymes.
    • Despite significant evolutionary divergence, the active site cleft formation and domain closure mechanisms remained largely unaffected in hybrids.
    • Enzyme activity was preserved in the hybrid constructs, suggesting functional robustness.

    Conclusions:

    • Evolutionary substitutions between human and yeast PGK do not substantially alter the enzyme's fundamental structure-function relationship.
    • The conserved nature of the active site and domain closure mechanism highlights functional redundancy or compensatory evolution.
    • Interspecies domain swapping is a viable method for probing enzyme structure-function dynamics and evolutionary conservation.