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Inhibition of a Zn(II)-containing enzyme, alcohol dehydrogenase, by anticancer antibiotics, mithramycin and chromomycin A3

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Abstract

One of the major attributes for the biological action of the aureolic acid anticancer antibiotics chromomycin A3 (CHR) and mithramycin (MTR) is their ability to bind bivalent cations such as Mg(II) and Zn(II) ions and form high affinity 2:1 complexes in terms of the antibiotic and the metal ion, respectively. As most of the cellular Zn(II) ion is found to be associated with proteins, we have examined the effect of MTR/CHR on the structure and function of a representative structurally well characterized Zn(II) metalloenzyme, alcohol dehydrogenase (ADH) from yeast. MTR and CHR inhibit enzyme activity of ADH with inhibitory constants of micromolar order. Results from size-exclusion column chromatography, dynamic light scattering, and isothermal titration calorimetry have suggested that the mechanism of inhibition of the metalloenzyme by the antibiotics is due to the antibiotic-induced disruption of the enzyme quaternary structure. The nature of the enzyme inhibition, the binding stoichiometry of two antibiotics per monomer, and comparable dissociation constants for the antibiotic and free (or substrate-bound) ADH imply that the association occurs as a consequence of the binding of the antibiotics to Zn(II) ion present at the structural center. Confocal microscopy shows the colocalization of the antibiotic and the metalloenzyme in HepG2 cells, thereby supporting the proposition of physical association between the antibiotic(s) and the enzyme inside the cell.

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Abbreviations

ADH:

Alcohol dehydrogenase

BSA:

Bovine serum albumin

CD:

Circular dichroism

CHR:

Chromomycin A3

DLS:

Dynamic light scattering

DPA:

Dipicolinic acid

HEPES:

4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid

ITC:

Isothermal titration calorimetry

MTR:

Mithramycin

NAD:

Nicotinamide adenine dinucleotide

NADH:

Reduced nicotinamide adenine dinucleotide

PBS:

Phosphate-buffered saline

Tris:

Tris(hydroxymethyl)aminomethane

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Acknowledgments

We thank Manabendra Mukherjee, Surface Physics Division, Saha Institute of Nuclear Physics, for allowing us to use the Zetasizer Nanoseries DLS instrument (Malvern Instruments, UK) and Mojammel Haque Mondal, Surface Physics Division, Saha Institute of Nuclear Physics, for his assistance during the DLS experiments. We also thank Swasti Raychaudhuri, Cyrstallography and Molecular Biology Division, Saha Institute of Nuclear Physics, for his assistance during the confocal microscopy experiments. We acknowledge CambridgeSoft for the free trial of the ChemDraw Pro 10.0 software package we used to draw the chemical structures.

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Authors and Affiliations

  1. Biophysics Division, Saha Institute of Nuclear Physics, Block-AF, Sector I, Bidhan Nagar, Kolkata, 700 064, India

    Pukhrambam Grihanjali Devi & Dipak Dasgupta

  2. Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Block-AF, Sector I, Bidhan Nagar, Kolkata, 700 064, India

    Prabir Kumar Chakraborty

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  1. Pukhrambam Grihanjali Devi
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Correspondence to Dipak Dasgupta.

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Devi, P.G., Chakraborty, P.K. & Dasgupta, D. Inhibition of a Zn(II)-containing enzyme, alcohol dehydrogenase, by anticancer antibiotics, mithramycin and chromomycin A3 . J Biol Inorg Chem 14, 347–359 (2009). https://doi.org/10.1007/s00775-008-0451-y

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