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Related Experiment Videos

Structure and function in yeast alcohol dehydrogenases.

B V Plapp, A J Ganzhorn, R M Gould

    Progress in Clinical and Biological Research
    |January 1, 1987
    PubMed
    Summary
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    Researchers compared Saccharomyces cerevisiae alcohol dehydrogenase (ADH) isozymes, mutagenizing ADH I to understand kinetic differences. Mutating specific amino acids revealed insights into enzyme function and substrate specificity, particularly concerning ethanol and longer-chain alcohols.

    Area of Science:

    • Biochemistry
    • Enzymology
    • Molecular Biology

    Background:

    • Saccharomyces cerevisiae possesses multiple alcohol dehydrogenase (ADH) isozymes (ADH I, II, III) with distinct kinetic properties.
    • Understanding these differences is crucial for elucidating enzyme function and substrate specificity.

    Purpose of the Study:

    • To compare the structure and kinetics of yeast ADH isozymes.
    • To investigate the role of specific amino acid residues in ADH I by site-directed mutagenesis.
    • To understand the basis for kinetic variations between ADH isozymes.

    Main Methods:

    • Construction of a yeast ADH model based on the homologous horse liver enzyme structure.
    • Steady-state kinetic studies at pH 7.3 and 30°C.
    • Site-directed mutagenesis of the ADH I gene to substitute key amino acid residues (e.g., Met-294 to Leu, His-47 to Arg, His-51 to Gln).

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    Main Results:

    • All studied isozymes follow the Ordered Bi Bi mechanism.
    • ADH II exhibits a 10-fold lower Michaelis constant for ethanol compared to ADH I and III.
    • Mutagenesis of Met-294 to Leu in ADH I did not replicate ADH II kinetics but enhanced activity with longer-chain alcohols.
    • Substitution of His-47 with Arg in ADH I decreased turnover numbers and altered coenzyme dissociation constants, conferring allyl alcohol resistance.
    • Replacing His-51 with Gln significantly reduced activity and abolished the pH-dependent kinetic profile.

    Conclusions:

    • Specific amino acid substitutions significantly impact yeast ADH kinetics and substrate preferences.
    • The His-47 residue is critical for coenzyme binding and catalytic efficiency, with implications for enzyme inhibition.
    • Further investigation is needed to fully explain the kinetic distinctions between ADH I and ADH II.