title: The metal ion requirements of Arabidopsis thaliana Glx2-2 for catalytic activity.
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 (1)H 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.
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA.
authors.forename: Pattraranee
authors.initials: P
authors.lastname: Limphong
authors.forename: Ross M
authors.initials: RM
authors.lastname: McKinney
authors.forename: Nicole E
authors.initials: NE
authors.lastname: Adams
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Makaroff
authors.forename: Brian
authors.initials: B
authors.lastname: Bennett
authors.forename: Michael W
authors.initials: MW
authors.lastname: Crowder
mesh_terms: D055162:Biocatalysis; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D009942:Organometallic Compounds / Q000737:chemistry* / Q000378:metabolism; D013869:Thiolester Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry*; D000099301:Hydroxyacylglutathione Hydrolase
chemical_list: D007501:Iron; D009942:Organometallic Compounds; D013869:Thiolester Hydrolases; D015032:Zinc; D000099301:Hydroxyacylglutathione Hydrolase