Document Type

Article

Language

eng

Format of Original

10 p.

Publication Date

2-2010

Publisher

Springer

Source Publication

Journal of Biological Inorganic Chemistry

Source ISSN

0949-8257

Original Item ID

doi: 10.1007/s00775-009-0593-6

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.

Comments

Accepted version. Journal of Biological Inorganic Chemistry, Vol. 15, No. 2 (February 2010): 249-258. DOI. © 2010 Springer. Used with permission.

Brian Bennett was affiliated with the Medical College of Wisconsin at the time of publication.

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