經酵母菌表現之綠竹筍苯丙胺酸脫氨裂解酶

Abstract

苯丙胺酸脫氨裂解酶 (phenylalanine ammonia lyase, EC 4.3.1.5，簡稱 PAL) 是植物 phenylpropanoids 生合成之第一個酵素。其催化苯丙胺酸 (L-phenylalanine) 進行非氧化性脫氨反應，產生肉桂酸 (trans-cinnamic acid)，再經由許多酵素催化，可衍生出許多的二級代謝物，如黃酮素、木質素等。 本論文為探討在 PAL 的催化反應中，可能有哪些胺酸參與，故以點突變的方式，改變特定胺酸，並利用嗜甲醇酵母菌 (Pichia pastoris) 來表現突變後的蛋白質。再將突變之蛋白質與野生型做生化性質比較，便能得知該胺酸在 PAL 催化反應中可能之角色。 三種表現蛋白質 YPAL 2、YPAL 2-SA 及YPAL 2-FH 之C 端帶有 (His)6-tag，可經由鎳離子親和管柱進行純化。經 10% SDS-PAGE 得知，三種表現蛋白質之單元體分子量皆約為 80 kD。表現蛋白質 YPAL 2 與 YPAL 2-FH 對基質 L-Phe 之 Km 值分別為 0.28 mM 與 1.11 mM；Vmax 分別為 0.043 與0.014 nkat。YPAL 2 最適反應溫度為 60℃，最適反應 pH 為 8.0，反應活化能為 11.8 kcal/mol；YPAL 2-FH 由於活性過低及酵素用盡，僅能完成基質飽和曲線實驗；YPAL 2-SA 則由於完全沒有活性，故無法進行生化性質之測定。 將 YPAL 2 活性區的關鍵胺酸由 serine 改為 alanine (S200A) 使得 PAL的活性完全消失，原因可能為：在失去 serine 的情況下，將導致活性區的反應基團 MIO 無法形成。而將活性區附近的 phenylalanine 改為 histidine (F397H)可能會影響反應中間產物之形成，故 PAL 活性降的十分的低。

Abstract Phenylalanine ammonia lyase (EC 4.3.1.5) is the first enzyme of the phenylpropanoid biosynthetic pathway. The enzyme catalyzes the non-oxidative deamination of L-phenylalanine to produce trans-cinnamic acid. This reaction leads to the biosynthesis of many phenylpropanoid derived compounds in plant such as flavonoids and lignin. In order to find out which amino acids play important roles in catalysis reaction of PAL, we use site-directed mutagenesis technique to change the specific amino acid residue of PAL and express it by the Pichia pastoris system. Three fusion protein YPAL 2、YPAL 2-SA and YPAL 2-FH containing C-terminal (His)6-tag was expressed then purified with Ni-column which retains proteins with polyhistidine tag. The subunit mass of the three expressed protein are about 80 kD. The Km values for phenylalanine are 0.28 mM for YPAL 2 and 1.11 mM for YPAL 2-FH. The Vmax values are 0.043 nkat for YPAL 2 and 0.014 nkat for YPAL 2-FH. The Kcat is 20.48 s-1 for YPAL 2 and 0.038 s-1 for YPAL 2-FH. The optimum temperature and pH for YPAL 2 are 60℃ and 8.0, respectively. The activation energy for YPAL 2 is 11.8 kcal/mol. Because of the amount and activity of YPAL 2-FH are very low we just can finish the substrate saturation experiment to deduce Km and Vmax values. In the other part, because the YPAL 2-SA totally loss its activity we can’t analyze its biochemical properties. By changing the amino acid in the active site of YPAL 2 from serine to alanine (S200A) could prevent the formation of MIO group which resulted in the losing of total activity. However, changing the amino acid near active site from phenylalanine to histidine (F397H) could affect the formation of intermediate and cause the decrease of PAL activity.