Strategic manipulation of the substrate specificity of Saccharomyces cerevisiae flavocytochrome b2

Simon N Daff, Forbes D C Manson, Graeme A Reid, Stephen K Chapman

    Research output: Contribution to journalArticle

    Abstract

    Flavocytochrome b(2) from Saccharomyces cerevisiae acts physiologically as an L-lactate dehydrogenase. Although L-lactate is its primary substrate, the enzyme is also able to utilize a variety of other (S)-2-hydroxy acids. Structural studies and sequence comparisons with several related flavoenzymes have identified the key active-site residues required for catalysis. However, the residues Ala-198 and Leu-230, found in the X-ray-crystal structure to be in contact with the substrate methyl group, are not well conserved. We propose that the interaction between these residues and a prospective substrate molecule has a significant effect on the substrate specificity of the enzyme. In an attempt to modify the specificity in favour of larger substrates, three mutant enzymes have been produced: A198G, L230A and the double mutant A198G/L230A. As a means of quantifying the overall kinetic effect of a mutation, substrate-specificity profiles were produced from steady-state experiments with (S)-2-hydroxy acids of increasing chain length, through which the catalytic efficiency of each mutant enzyme with each substrate could be compared with the corresponding wild-type efficiency. The Ala-198-->Gly mutation had little influence on substrate specificity and caused a general decrease in enzyme efficiency. However, the Leu-230-->Ala mutation caused the selectivity for 2-hydroxyoctanoate over lactate to increase by a factor of 80.

    Original languageEnglish
    Pages (from-to)829-834
    Number of pages6
    JournalBiochemical Journal
    Volume301
    Issue number3
    Publication statusPublished - 1 Aug 1994

    Keywords

    • ESCHERICHIA-COLI
    • SEQUENCE
    • ELECTRON-TRANSFER
    • OXIDASE
    • DEHYDROGENASE
    • BAKERS-YEAST
    • CYTOCHROME B2
    • MOLECULAR WEIGHT
    • EXPRESSION
    • MUTAGENESIS

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