甘藷塊根澱粉磷解脢不需醣引子活性之分子機制探討

Abstract

中文摘要 本研究發現，甘藷塊根L型澱粉磷解脢 (L-SP) 中央插入序列 (L78) 斷裂後，不需醣引子的活性 (primer-independent activity, PI activity) 便會消失，如果外加表現的L78於斷裂完全的L-SP中，可使PI活性恢復。以電腦軟體Clustal_W將L-SP與NDP-Glc pryophosphorylases (NDPG PPase) 進行序列比對，結果發現NDPG PPase與Glc-1-P產生交互作用的兩個胺基酸，也出現在L-SP上的Glu528以及Lys529。再以電腦程式Discovery Studio (ver. 1.7) 分析根據兔肌GP所模擬出來的L-SP結構，發現於L78上有兩個可能的結合口袋，且其周圍繞著三組Glu及Lys，其中一組就是Glu528及Lys529，以上證據顯示L78可能為PI activity提供Glc-1-P結合區 (B site)。PI activity隨著反應時間增加，出現了三相催化反應 (initiation phase、elongation phase以及steady-state phase)。本實驗利用外加G1~G7 (G1為葡萄糖)，以模擬反應中逐漸延長的直鏈短醣，經酵素動力學檢視，發現L-SP在外加G4以上的直鏈短醣時，有最高催化效率，因此推測G4可能為initiation phase 進入elongation phase的轉折點。另外L-SP無論斷裂與否，都可以使用G2以上的直鏈短醣作為基質，因此推論L-SP所需要的最短primer應該為G2。最後，實驗結果顯示PI activity的催化行為，具有異位調控性質，而Glc與ADPGlc分別是其正效應物與負效應物。本論文結果顯示L-SP在澱粉合成的起始步驟，可能扮演重要的角色。

Abstract When the 78-amino acid insertion (L78) in the middle of the L-form starch phosphorylase molecule was removed, the primer-independent activity (PI activity) was lost completely. However, the addition of extrinsic expressed L78 could rescue the PI activity. The sequence alignment of L-SP and NDP-Glc pyrophosphorylases (PPase) was generated using the computer program Clustal_W. The two-residue pair (Glu, Lys) that appear to interact with Glc-1-P in NDPG PPases was conserved in the L78 (Glu528, Lys529). The structure of L-SP derived from comparative modeling using the coordinates of the rabbit muscle glycogen phosphorylase as the template was analyzed by Discovery Studio (ver. 1.7), and two binding pockets were predicted in the L78. There were three groups of the Glu-Lys repeat surrounding these binding pockets, including Glu528 and Lys529. These observations suggested that L78 might serve as the second Glc-1-P binding site (B site) for the conjugation with the first Glc-1-P in the active site (A site). PI activity showed three catalytic phases (initiation phase, elongation phase and steady-state phase) during the PI catalytic reaction (Chen, 1997). The kinetic parameters of L-SP were observed by using several maltosaccharides (G1~G7, G1 is the glucose) to simulate the synthetic reaction of PI activity. G4 or longer showed higher catalytic effect, indicating that G4 might be the favored substrate for elongation, and served as a turning point to bring the initiation phase to the elongation phase. The G2 was the smallest primer for L-SP, although it was not an effective substrate. In addition, we found that L-SP was allosterically controlled in its PI activity, in which Glc and ADPG served as positive and negative effectors, respectively. The results in this study suggested that L-SP could be involved in the primer synthesis for starch formation.