The metal ion requirements of Arabidopsis thaliana Glx2-2 for catalytic activity.
The metal ion requirements of Arabidopsis thaliana Glx2-2 for catalytic activity.
复制标题
拟南芥 Glx2-2 催化活性对金属离子的需求。
DOI：
10.1007/s00775-009-0593-6
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发表时间：
2010
期刊：
影响因子：
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通讯作者：
Crowder,MichaelW
中科院分区：
文献类型：
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作者：
Limphong,Pattraranee;McKinney,RossM;Adams,NicoleE;Makaroff,ChristopherA;Bennett,Brian;Crowder,MichaelW
为了更好地理解拟南芥Glx 2 -2的结构、金属含量、金属中心的性质和酶活性，该酶被过表达、纯化，并使用金属分析、动力学、UV-vis、EPR和1H NMR光谱表征。在纯化过程中分离紫色、黄色或无色的含Glx 2 -2的级分，发现不同颜色的级分含有不同量的Fe和Zn（II）。光谱分析的离散馏分提供的证据Fe（II），Fe（III），Fe（III）-Zn（II），和反铁磁耦合的Fe（II）-Fe（III）中心分布在离散的Glx 2 -2-含馏分。个别的稳态动力学常数之间的分馏物种，取决于存在的金属离子的数量和类型。然而，有趣的是，催化效率常数，kcat/Km，是不变的馏分之间。kcat/Km的值在低的生理底物浓度下控制催化速率。我们认为，独立ofkcat/Kmon的精确构成的活性位点金属中心是进化相关的缺乏选择性的Fe与Zn（II）或Fe（II）与Fe（III），在一个或多个金属结合位点。
In an effort to better understand the structure, metal content, the nature of the metal centers, and enzyme activity ofArabidopsis thalianaGlx2-2, the enzyme was overexpressed, purified, and characterized using metal analyses, kinetics, and UV–vis, EPR, and1H 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 ofkcat/Kmgoverns the catalytic rate at low, physiological substrate concentrations. We suggest that the independence ofkcat/Kmon 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.