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The metal ion requirements of Arabidopsis thaliana Glx2-2 for catalytic activity

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Abstract

In an effort to better understand the structure, metal content, the nature of the metal centers, and enzyme activity of Arabidopsis thaliana Glx2-2, the enzyme was overexpressed, purified, and characterized using metal analyses, kinetics, and UV–vis, EPR, and 1H NMR spectroscopies. Glx2-2-containing fractions that were purple, yellow, or colorless were separated during purification, and the differently colored fractions were found to contain different amounts of Fe and Zn(II). Spectroscopic analyses of the discrete fractions provided evidence for Fe(II), Fe(III), Fe(III)–Zn(II), and antiferromagnetically coupled Fe(II)–Fe(III) centers distributed among the discrete Glx2-2-containing fractions. The individual steady-state kinetic constants varied among the fractionated species, depending on the number and type of metal ion present. Intriguingly, however, the catalytic efficiency constant, k cat/K m, was invariant among the fractions. The value of k cat/K m governs the catalytic rate at low, physiological substrate concentrations. We suggest that the independence of k cat/K m on the precise makeup of the active-site metal center is evolutionarily related to the lack of selectivity for either Fe versus Zn(II) or Fe(II) versus Fe(III), in one or more metal binding sites.

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Abbreviations

FPLC:

Fast performance liquid chromatography

Glx1:

Lactoylglutathione lyase

Glx2:

Hydroxyacylglutathione hydrolase

MOPS:

3-(N-Morpholino)propanesulfonic acid

SLG:

S-(2-Hydroxyacyl)glutathione

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Acknowledgments

This work was supported by the National Institutes of Health (AI056231 to B.B., GM076199-01A2 to C.A.M., and EB001980 to the Medical College of Wisconsin), a Miami University/Volwiler Professorship (to M.W.C), and an Presidential Academic Enrichment fellowship (to P.L.).

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Miami University, 160 Hughes Hall, Oxford, OH, 45056, USA

    Pattraranee Limphong, Ross M. McKinney, Nicole E. Adams, Christopher A. Makaroff & Michael W. Crowder

  2. Department of Biophysics, National Biomedical EPR Center, Medical College of Wisconsin, Milwaukee, WI, 53226, USA

    Brian Bennett

Authors
  1. Pattraranee Limphong
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  2. Ross M. McKinney
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  3. Nicole E. Adams
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  4. Christopher A. Makaroff
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  5. Brian Bennett
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  6. Michael W. Crowder
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Correspondence to Michael W. Crowder.

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Limphong, P., McKinney, R.M., Adams, N.E. et al. The metal ion requirements of Arabidopsis thaliana Glx2-2 for catalytic activity. J Biol Inorg Chem 15, 249–258 (2010). https://doi.org/10.1007/s00775-009-0593-6

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  • DOI: https://doi.org/10.1007/s00775-009-0593-6

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