Original Paper

JBIC Journal of Biological Inorganic Chemistry

, Volume 15, Issue 2, pp 249-258

First online:

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

  • Pattraranee LimphongAffiliated withDepartment of Chemistry and Biochemistry, Miami University
  • , Ross M. McKinneyAffiliated withDepartment of Chemistry and Biochemistry, Miami University
  • , Nicole E. AdamsAffiliated withDepartment of Chemistry and Biochemistry, Miami University
  • , Christopher A. MakaroffAffiliated withDepartment of Chemistry and Biochemistry, Miami University
  • , Brian BennettAffiliated withDepartment of Biophysics, National Biomedical EPR Center, Medical College of Wisconsin
  • , Michael W. CrowderAffiliated withDepartment of Chemistry and Biochemistry, Miami University Email author 

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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.


Glyoxalase 2 Metalloenzyme Iron Zinc Catalysis