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NADP-malic enzyme from maize leaves: a fluorescence study

M F Drincovich1, C S Andreo

  • 1Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), (CONICET, Fund M Lillo, Universidad Nacional de Rosario), Argentina.

Biochemistry and Molecular Biology International
|August 1, 1995
PubMed
Summary
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Maize NADP-malic enzyme was labeled with eosin-5-maleimide (EMA). This probe identified sulfhydryl groups within the nicotinamide adenine dinucleotide phosphate (NADP) binding site, crucial for enzyme activity.

Area of Science:

  • Biochemistry
  • Enzymology
  • Plant Molecular Biology

Background:

  • NADP-malic enzyme (NADP-ME) is vital in plant carbon metabolism.
  • Understanding the NADP-binding site is key to elucidating enzyme regulation.
  • Fluorescent probes offer sensitive methods for studying enzyme active sites.

Purpose of the Study:

  • To identify the specific residues modified by eosin-5-maleimide (EMA) in maize NADP-malic enzyme.
  • To investigate the structural and functional relationship between the NADP-binding site and reactive sulfhydryl groups.
  • To characterize the interaction of NADP and Mg2+ with the enzyme's fluorescent properties.

Main Methods:

  • Covalent labeling of maize NADP-malic enzyme with eosin-5-maleimide (EMA).
  • Spectroscopic analysis, including emission fluorescence spectra and intrinsic fluorescence quenching.

Related Experiment Videos

  • Extrinsic fluorescence quenching studies to probe the NADP-binding site.

    • Main Results:

    • EMA specifically labeled sulfhydryl groups protected by NADP.
    • Fluorescence studies indicated proximity between native enzyme's tryptophanyl groups and EMA-modified sites.
    • NADP binding enhanced intrinsic fluorescence, an interaction strengthened by Mg2+.
    • EMA modification of the NADP-binding site prevented further nucleotide interaction.

    Conclusions:

    • Eosin-5-maleimide (EMA) covalently labels sulfhydryl groups within the NADP-binding site of maize NADP-malic enzyme.
    • These identified sulfhydryl groups are critical for NADP binding and enzyme function.
    • The study provides insights into the structural organization of the NADP-binding site.