Transglutaminase 2 (TGase2) is involved in a variety of cellular processes and diseases via its transamidase and kinase activities, which are regulated by conformational changes induced by the binding of nucleotides and divalent cations. However, due to the lack of an appropriate assay system, the function of critical amino acid residues in the regulation of both activities is unclear. Thus, we designed site-directed TGase2 mutants that were then used in protein arrays to investigate the effects of the mutations on the regulation of TGase2 transamidase and kinase activities. We found that the Lys444Ala mutation, but not the Arg580Lys and Lys663Ala mutations, completely inhibited the transamidase activity. Additionally, the mutations at Lys444 and Lys663 inhibited the kinase activity by 27% and 48%, respectively, but the mutations at Cys277 and Arg580 had no effect. Furthermore, a kinetic analysis of the transamidation reaction revealed that the Lys663Ala mutation increased the affinity of TGase2 for the substrate fibrinogen. Thus, this array-based approach would be helpful for investigation of amino acids responsible for regulation of the TGase2 transamidase and kinase activities and the pathogenesis of TGase2- mediated diseases.

中文翻译：

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基于阵列的氨基酸负责转氨酶和转谷氨酰胺酶2激酶活性的调控。

转谷氨酰胺酶2（TGase2）通过其转酰胺酶和激酶活性参与多种细胞过程和疾病，这些活性受核苷酸和二价阳离子结合诱导的构象变化的调节。然而，由于缺乏合适的测定系统，关键氨基酸残基在两种活性调节中的功能尚不清楚。因此，我们设计了定点的TGase2突变体，然后将其用于蛋白质阵列中，以研究突变对TGase2转酰胺酶和激酶活性的调节作用。我们发现Lys444Ala突变，但不是Arg580Lys和Lys663Ala突变，完全抑制了转酰胺酶的活性。此外，Lys444和Lys663处的突变分别将激酶活性抑制了27％和48％，但Cys277和Arg580处的突变无效。此外，对转酰胺基反应的动力学分析表明，Lys663Ala突变增加了TGase2对底物纤维蛋白原的亲和力。因此，这种基于阵列的方法将有助于研究负责调节TGase2转酰胺酶和激酶活性的氨基酸以及TGase2介导的疾病的发病机理。