磷酸化修飾對甘藷塊根 L 型澱粉磷解脢之影響

Abstract

轉譯後修飾 (post-translational modifications) 乃生物細胞調控蛋白質功能的主要方式。目前已知甘藷塊根L型澱粉磷解脢 (L-SP) 會受到磷酸化 (楊光華,2005) 及蛋白裂解修飾 (陳翰民,1997)，而本論文即探討這兩種修飾對L-SP活性之影響。磷酸化修飾並未明顯影響L-SP需醣引子合成澱粉及磷解澱粉之活性，但是可加速L-SP中央L78降解的速率。所以磷酸化修飾可能是加速降解L78的訊號，而並非是影響L-SP催化效率的調控方式。L78降解後，由需醣引子合成澱粉活性之動力學結果發現，L-SP對可溶性澱粉的親和力增加，對Glc-1-P親和力則小幅下降，但未顯著影響L-SP催化能力。過去本實驗室認為L78的降解可能是將L-SP的活性，自合成澱粉方向轉變為磷解澱粉之角色的調控方式，但由本論文觀察L-SP磷解澱粉之動力學結果，發現L-SP對可溶性澱粉的親和力依然提升，對磷酸親和力不變，也不會明顯影響L-SP磷解澱粉的活性。 另外，探討L-SP不需醣引子合成直鏈醣活性時，發現磷酸化修飾及L78移除會明顯降低其催化效率，令我們認為L-SP催化不需醣引子合成直鏈醣的活性區，與催化需醣引子合成澱粉及磷解澱粉的活性區不同。同時，我們推測L78可能就是催化不需醣引子合成直鏈醣的活性區。

Post-translational modification is a major mechanism regulating protein functions in the eukaryote. We have found that L-SP in sweet potato roots can be modified by phosphorylation and proteolytic cleavage at specific sites. In this study, we tried to explore the effects of these two modifications on L-SP. According to the kinetic studies, phosphorylation of L-SP has no effect on its catalytic behavior either in primer-dependent synthetic or phosphorolytic activity, but increases the sensitivity for the proteolysis of L78 on L-SP. Enzyme kinetic studies of L-SP in primer-dependent synthesis indicates that the affinity toward soluble starch increases after the proteolytic modification of L78. However, the affinity toward Glc-1-P decreases, and shows no obvious changes in its catalytic efficiency in terms of kcat/Km. According to our previous studies, we expected that the proteolysis on L78 may switch the catalytic direction of L-SP from starch synthesis to starch phosphorolysis. Nevertheless, results in this study showed that the affinity to soluble starch increases after the proteolytic modification of L78, whereas the affinity toward Pi and the catalytic efficiency of phosphorylation keep unchanged. When we looked forward into the primer-independent synthetic activity of L-SP, we found that phosphorylation and proteolytic modification would decrease the catalytic efficiency. This phenomenon might be resulted from the different active sites for catalyzing primer-dependent and primer-independent synthetic reaction. Thus, we supposed L78 is the catalytic site of primer-independent synthesis process.