Frueauf Jeremiah B, Ballicora Miguel A, Preiss Jack
Department of Biochemistry and Molecular Biology, Michigan State University, Biochemistry Building, East Lansing, MI 48824, USA.
Plant J. 2003 Feb;33(3):503-11. doi: 10.1046/j.1365-313x.2003.01643.x.
Asp142 in the homotetrameric ADP-glucose pyrophosphorylase (ADP-Glc PPase) enzyme from Escherichia coli was demonstrated to be involved in catalysis of this enzyme [Frueauf, J.B., Ballicora, M.A. and Preiss J. (2001) J. Biol. Chem., 276, 46319-46325]. The residue is highly conserved throughout the family of ADP-Glc PPases, as well as throughout the super-family of sugar-nucleotide pyrophosphorylases. In the heterotetrameric ADP-Glc PPase from potato (Solanum tuberosum L.) tuber, the homologous residue is present in both the small (Asp145) and the large (Asp160) subunits. It has been proposed that the small subunit of plant ADP-Glc PPases is catalytic, while the large subunit is modulatory; however, no catalytic residues have been identified. To investigate the function of these conserved Asp residues in the ADP-Glc PPase from potato tuber, we used site-directed mutagenesis to introduce either an Asn or a Glu. Kinetic analysis in the direction of synthesis or pyrophosphorolysis of ADP-Glc showed a significant decrease (more than four orders of magnitude) in the specific activity of the SD145NLwt, SD145NLD160N, and SD145NLD160E mutants, while the effect was smaller (approximately two orders of magnitude) with the SD145ELwt, SD145ELD160N, and SD145ELD160E mutants. By contrast, mutation of the large subunit alone did not affect the specific activity but did alter the apparent affinity for the activator 3-phosphoglycerate, showing two types of apparent roles for this residue in the different subunits. These results show that mutation of Asp160 of the large subunit does not affect catalysis, thus the large subunit is not catalytic, and that the negative charge of Asp145 in the small subunit is necessary for enzyme catalysis.
已证明来自大肠杆菌的同源四聚体 ADP - 葡萄糖焦磷酸化酶（ADP - Glc PPase）中的 Asp142 参与该酶的催化作用[弗鲁奥夫，J.B.，巴利科拉，M.A.和普赖斯 J.（2001 年）《生物化学杂志》，276，46319 - 46325]。该残基在整个 ADP - Glc PPase 家族以及整个糖核苷酸焦磷酸化酶超家族中高度保守。在马铃薯（Solanum tuberosum L.）块茎的异源四聚体 ADP - Glc PPase 中，同源残基存在于小亚基（Asp145）和大亚基（Asp160）中。有人提出植物 ADP - Glc PPase 的小亚基具有催化作用，而大亚基具有调节作用；然而，尚未鉴定出催化残基。为了研究马铃薯块茎 ADP - Glc PPase 中这些保守 Asp 残基的功能，我们使用定点诱变引入 Asn 或 Glu。对 ADP - Glc 的合成或焦磷酸解方向的动力学分析表明，SD145NLwt、SD145NLD160N 和 SD145NLD160E 突变体的比活性显著降低（超过四个数量级），而 SD145ELwt、SD145ELD160N 和 SD145ELD160E 突变体的影响较小（约两个数量级）。相比之下，仅大亚基的突变不影响比活性，但确实改变了对激活剂 3 - 磷酸甘油酸的表观亲和力，表明该残基在不同亚基中具有两种明显的作用。这些结果表明，大亚基 Asp160 的突变不影响催化作用，因此大亚基不具有催化作用，并且小亚基中 Asp145 的负电荷对于酶催化是必需的。
