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LDH-C4-substrate binary complexes studied by intrinsic fluorescence method

G S Gupta1, B P Kang

  • 1Department of Biophysics, Panjab University, Chandigarh, India.

Indian Journal of Biochemistry & Biophysics
|June 1, 1997
PubMed
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Lactate dehydrogenase-C4 (LDH-C4) interactions with NAD+, NADH, pyruvate, and lactate were studied using fluorescence. NAD+ and NADH significantly quenched LDH-C4 fluorescence, indicating strong binding, while pyruvate and lactate showed weaker interactions.

Area of Science:

  • Biochemistry
  • Enzymology
  • Spectroscopy

Background:

  • Lactate dehydrogenase-C4 (LDH-C4) is an enzyme crucial in metabolic pathways.
  • Understanding LDH-C4's substrate binding is essential for elucidating its catalytic mechanisms.
  • Intrinsic fluorescence offers a sensitive method to probe enzyme-ligand interactions.

Purpose of the Study:

  • To investigate the binding interactions of LDH-C4 with its substrates and cofactors.
  • To quantify the binding affinities using fluorescence spectroscopy.
  • To characterize the conformational changes in LDH-C4 upon ligand binding.

Main Methods:

  • Intrinsic fluorescence measurements of LDH-C4.
  • Excitation and emission spectra analysis.
  • Quenching studies with NAD+, NADH, pyruvate, and lactate.

Related Experiment Videos

  • Determination of association constants (Ka) and Stern-Volmer constants (Ksv).

    • Main Results:

    • LDH-C4 exhibited excitation and emission maxima at 282 nm and 340 nm, respectively.
    • NAD+ and NADH caused significant fluorescence quenching (92-93%), indicating strong binding.
    • Pyruvate and lactate showed lower quenching (29% and 21%), with biphasic Stern-Volmer plots suggesting complex binding mechanisms.
    • Association constants (Ka) varied, with NADH showing the highest (20 x 10^4 M⁻¹).
    • Red shifts in emission spectra upon lactate and pyruvate binding indicated exposure of buried chromophores.

    Conclusions:

    • LDH-C4 displays distinct binding behaviors with its cofactors (NAD+, NADH) and substrates (pyruvate, lactate).
    • Fluorescence quenching and spectral shifts provide insights into the enzyme's conformational dynamics during substrate interaction.
    • The findings contribute to a deeper understanding of LDH-C4's structure-function relationship.