JBIC Journal of Biological Inorganic Chemistry

, Volume 15, Issue 2, pp 249–258

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

  • Pattraranee Limphong
  • Ross M. McKinney
  • Nicole E. Adams
  • Christopher A. Makaroff
  • Brian Bennett
  • Michael W. Crowder
Original Paper

DOI: 10.1007/s00775-009-0593-6

Cite this article as:
Limphong, P., McKinney, R.M., Adams, N.E. et al. J Biol Inorg Chem (2010) 15: 249. doi:10.1007/s00775-009-0593-6
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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, kcat/Km, was invariant among the fractions. The value of kcat/Km governs the catalytic rate at low, physiological substrate concentrations. We suggest that the independence of kcat/Km 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.

Keywords

Glyoxalase 2MetalloenzymeIronZincCatalysis

Abbreviations

FPLC

Fast performance liquid chromatography

Glx1

Lactoylglutathione lyase

Glx2

Hydroxyacylglutathione hydrolase

MOPS

3-(N-Morpholino)propanesulfonic acid

SLG

S-(2-Hydroxyacyl)glutathione

Copyright information

© SBIC 2009

Authors and Affiliations

  • Pattraranee Limphong
    • 1
  • Ross M. McKinney
    • 1
  • Nicole E. Adams
    • 1
  • Christopher A. Makaroff
    • 1
  • Brian Bennett
    • 2
  • Michael W. Crowder
    • 1
  1. 1.Department of Chemistry and BiochemistryMiami UniversityOxfordUSA
  2. 2.Department of BiophysicsNational Biomedical EPR Center, Medical College of WisconsinMilwaukeeUSA