A novel assay method for an amino acid racemase reaction based on circular dichroism
A novel assay method for an amino acid racemase reaction based on circular dichroism
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DOI：
10.1042/bj20041649
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发表时间：
2005-07-15
影响因子：
4.1
通讯作者：
Sugiyama, M
中科院分区：
文献类型：
--
作者：
Noda, M;Matoba, Y;Sugiyama, M
我们建立了一种基于圆二色性的新型测定方法，可用于氨基酸消旋酶（例如ALR（丙氨酸消旋酶））活性的动力学研究。尽管酶偶联测定方法已用于测量消旋酶活性，但 CD 方法优于酶测定，因为它可以准确确定对映异构体在其 L 型和 D 型之间消旋化时的直接变化。酶联检测需要 D-氨基酸氧化酶，该酶可被 ALR 抑制剂 D-环丝氨酸灭活。这表明酶偶联测定法对D-环丝氨酸对ALR的抑制动力学研究仅限于分析D-Ala生成L-Ala的反应，而CD测定法不需要偶联酶，因此可以综合评价L-Ala生成D-Ala和Of的反应。 在多种底物浓度下，从 D-Ala 中提取 L-Ala。链霉菌 ALR 还催化 L-Ser 形成 D-Ser 和 D-Ser 形成 L-Ser，但催化常数 (k(cat)) 分别比 L-Ala 形成 D-Ala 和 D-Ala 形成 L-Ala 的催化常数低 4 倍和 10 倍。
We have established a novel assay method based on circular dichroism that can be used for the kinetic study of the activity of amino acid racemases, such as ALR (alanine racemase). Although an enzyme-coupled assay method has been used to measure racemase activity, the CD method is superior to the enzyme assay because it can accurately determine the immediate changes of an enantiomer on racemization between its L- and D-forms. The enzyme-coupled assay requires D-amino acid oxidase, which is inactivated by an inhibitor of ALR, D-cycloserine. This indicates that the inhibitory kinetic study for ALR with D-cycloserine by the enzyme-coupled assay method is restricted to the analysis of only the reaction resulting in the formation Of L-Ala from D- Ala. However, since the CD assay does not require the coupled enzyme, it can be used to comprehensively evaluate the reactions that result in the formation both Of D-Ala from L-Ala and Of L-Ala from D-Ala at several substrate concentrations. Streptomyces ALR also catalyses the formation of D-Ser from L-Ser and of L-Ser from D-Ser, but the catalytic constants (k(cat))are 4- and 10-fold lower than those for the formation of D-Ala from L-Ala and of L-Ala from D-Ala respectively.