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
    • Department of Chemistry and BiochemistryMiami University
  • Ross M. McKinney
    • Department of Chemistry and BiochemistryMiami University
  • Nicole E. Adams
    • Department of Chemistry and BiochemistryMiami University
  • Christopher A. Makaroff
    • Department of Chemistry and BiochemistryMiami University
  • Brian Bennett
    • Department of BiophysicsNational Biomedical EPR Center, Medical College of Wisconsin
    • Department of Chemistry and BiochemistryMiami University
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


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.


Glyoxalase 2MetalloenzymeIronZincCatalysis



Fast performance liquid chromatography


Lactoylglutathione lyase


Hydroxyacylglutathione hydrolase


3-(N-Morpholino)propanesulfonic acid



Copyright information

© SBIC 2009