The amine transaminase from Chromobacterium violaceum (Cv-ATA) is a pyridoxal-5'-phosphate (PLP) dependent enzyme. The biological activity of this enzyme requires the formation of a holo homo dimer. The operational stability of Cv-ATA is, however, low due to dimer dissociation. At the enzyme dimeric interface, two phosphate group binding cups (PGBC) are located. Each cup coordinates the phosphate group of PLP by hydrogen bonds originating from both subunits. Hypothetically, molecular coordination of phosphate groups (PLP or free inorganic phosphate) into the PGBC can affect both dimer stabilization and enzyme activity. To test this assumption, the influence of phosphate (as a functional group in PLP or as free inorganic anions) on the stability and activity of Cv-ATA was explored by various biophysical techniques. The results show that Cv-ATA has a relatively low affinity towards PLP, which results in an excess of apo dimeric enzyme after enzyme purification. Incubation of the apo dimer in buffer solution supplemented with PLP restored the active holo dimer. The addition of PLP or inorganic phosphate into the enzyme storage solutions protected Cv-ATA from both chemical and long term storage unfolding. The use of phosphate buffer leads to faster inactivation of the holo enzyme, compared to the use of HEPES buffer. These results open up for new perspectives on how to improve the stability of PLP-dependent enzymes.

来自紫色杆菌（Cv- ATA）的胺转氨酶是一种吡pyr醛-5'-磷酸（PLP）依赖性酶。该酶的生物学活性需要形成完整的同源二聚体。Cv的操作稳定性然而，由于二聚体解离，-ATA低。在酶二聚体界面处，定位了两个磷酸基团结合杯（PGBC）。每个杯通过源自两个亚基的氢键配位PLP的磷酸基团。假设地，进入PGBC的磷酸基团（PLP或游离的无机磷酸酯）的分子配位会影响二聚体的稳定和酶的活性。为了验证该假设，通过各种生物物理技术探索了磷酸盐（作为PLP中的官能团或作为游离无机阴离子）对Cv -ATA稳定性和活性的影响。结果表明，Cv-ATA对PLP的亲和力较低，导致酶纯化后载脂蛋白二聚体酶过量。在补充了PLP的缓冲溶液中孵育载脂蛋白二聚体可恢复活性的完整二聚体。在酶存储溶液中添加PLP或无机磷酸盐可保护Cv -ATA免受化学和长期存储的影响。与使用HEPES缓冲液相比，使用磷酸盐缓冲液可以更快地灭活全酶。这些结果为如何改善PLP依赖性酶的稳定性开辟了新的前景。