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Metal binding to D-lactate dehydrogenase

F F Morpeth, V Massey

    Biochemistry
    |March 16, 1982
    PubMed
    Summary

    Cobalt-substituted D-lactate dehydrogenase shows high-spin tetrahedral cobalt properties and is inhibited by D-lactate. Its kinetics suggest metal involvement in substrate binding and reduction, with a second metal-binding site identified.

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    Biochemistry·2001

    Area of Science:

    • Biochemistry
    • Enzymology
    • Bioinorganic Chemistry

    Background:

    • D-lactate dehydrogenase (LDH) is a crucial enzyme in metabolic pathways.
    • Understanding the role of metal cofactors in LDH function is essential for elucidating its catalytic mechanism.

    Purpose of the Study:

    • To compare the kinetic and spectral properties of native (zinc) and cobalt-substituted D-lactate dehydrogenase.
    • To investigate the role of the metal cofactor in substrate binding and reduction.
    • To characterize a potential second metal-binding site.

    Main Methods:

    • Spectroscopic analysis including Optical and Magnetic Circular Dichroism (MCD).
    • Kinetic studies of enzyme activity and inhibition.
    • Enzyme assays with varying substrates and electron acceptors.

    Main Results:

    • Optical and MCD spectra indicate high-spin tetrahedral cobalt in the substituted enzyme.
    • Cobalt-LDH exhibits strong inhibition by D-lactate with oxygen as the electron acceptor, suggesting abortive complex formation.
    • Substrate reduction follows a triphasic pattern involving a transient charge-transfer complex, similar to the native enzyme.
    • Kinetic data imply metal involvement in substrate binding and reduction.
    • Evidence for a second metal-binding site involving flavin and an essential thiol was found.

    Conclusions:

    • Cobalt substitution provides insights into the active site metal's role in D-lactate dehydrogenase catalysis.
    • The enzyme's mechanism involves metal-dependent substrate binding and reduction.
    • A secondary metal-binding site, potentially involving flavin and thiol residues, plays a role in enzyme function.

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