Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix Hausdorff-Strasse 4, 17487 Greifswald, Germany.
Anal Chem. 2010 Mar 1;82(5):2082-6. doi: 10.1021/ac9028483.
Amine-transaminases (ATAs, omega-transaminases, omega-TA) are PLP-dependent enzymes that catalyze amino group transfer reactions. In contrast to the widespread and well-known amino acid-transaminases, ATAs are able to convert substrates lacking an alpha-carboxylic functional group. They have gained increased attention because of their potential for the asymmetric synthesis of optically active amines, which are frequently used as building blocks for the preparation of numerous pharmaceuticals. Having already introduced a fast kinetic assay based on the conversion of the model substrate alpha-methylbenzylamine for the characterization of the amino acceptor specificity, we now report on a kinetic conductivity assay for investigating the amino donor specificity of a given ATA. The course of an ATA-catalyzed reaction can be followed conductometrically since the conducting substrates, a positively charged amine and a negatively charged keto acid, are converted to nonconducting products, a noncharged ketone and a zwitterionic amino acid. The decrease of conductivity for the investigated reaction systems were determined to be 33-52 microS mM(-1). In contrast to other ATA-assays previously described, with this approach all transamination reactions between any amine and any keto acid can be monitored without the need for an additional enzyme or staining solutions. The assay was used for the characterization of a ATA from Rhodobacter sphaeroides, and the data obtained were in excellent agreement with gas chromatography analysis.
胺基转移酶（ATAs，ω-转氨酶，ω-TA）是依赖于 PLP 的酶，可催化氨基转移反应。与广泛存在且广为人知的氨基酸转氨酶不同，ATAs 能够转化缺乏α-羧基官能团的底物。由于它们在立体选择性合成光学活性胺方面的潜力，它们受到了越来越多的关注，这些胺经常被用作制备许多药物的构建块。我们已经引入了一种基于模型底物α-甲基苄基胺转化的快速动力学测定法来表征氨基酸接受体特异性，现在我们报告了一种用于研究给定 ATA 的氨基酸供体特异性的动力学电导率测定法。由于导电底物，即带正电荷的胺和带负电荷的酮酸，被转化为非导电产物，即非电荷酮和两性离子氨基酸，因此可以通过电导率法跟踪 ATA 催化的反应过程。对于所研究的反应体系，电导率的降低被确定为 33-52 μS mM(-1)。与以前描述的其他 ATA 测定方法不同，通过这种方法可以监测任何胺和任何酮酸之间的所有转氨反应，而无需额外的酶或染色溶液。该测定法用于表征来自 Rhodobacter sphaeroides 的 ATA，并且获得的数据与气相色谱分析非常吻合。
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