白蘆筍苯丙胺酸裂解酶之生化學研究

Abstract

苯丙胺酸裂解酶 (phenylalanine ammonia lyase, PAL EC 4.3.1.5) 為植物體內 phenylpropanoid 代謝路徑中的第一個反應酵素。可催化 L-phenylalanine 行脫氨反應成 trans-cinnamic acid 和 氨，以促使植物二級代謝產物及大量 phenylpropanoids 衍生物生合成，包括木質素 (lignin)、黃酮素 (flavonoid) 等。本論文以白蘆筍嫩莖為材料，經緩衝液粗抽、硫酸銨分劃、膠體過濾法 (Sepharacyl S-300)、疏水性作用層析法 (Phenyl-Sepharose) 及快速蛋白質液相層析法 (FPLC) 後，最後得到純化倍率為 65 倍，比活性為 41 nkat/mg protein。以 FPLC Superose 12 測得 PAL 原態分子量約 288 kD，次單元體分子量由 SDS-PAGE 測得為約 75 kD，因此推測綠竹 PAL 可能為四元體結構。利用綠竹免疫染色法之專一性特性，顯示純化所得結果確實為 PAL。 白蘆筍 PAL 之生化性質，最適反應溫度為 55℃，Km 值為 982 μM，活化能為 22.5 kcal/mol，最適反應 pH 為 8.5。酵素活性會受到鎂、鈣離子的活化；錳、鈷、汞離子的抑制。二級代謝產物，如 trans-cinnamic acid, p-amino-benzonic acid, caffeic acid 及 tannic acid 對 PAL 有回饋抑制現象。PAL 對基質 L-phenylalanine 有專一性。酵素催化活化區域具有 serine、tyrosine、histidine 胺酸。

Phenylalanine ammonia lyase (PAL, EC 4.3.1.5) is the first enzyme of the phenylpropanoid metabolic pathway. The enzyme catalyzes the deamination of L-phenylalanine to form trans-cinnamic acid and ammonia. This reaction leads to biosynthesis of many phenylpropanoid-derived secondary metabolic products in plants, such as flavonoids and lignin. In this study, PAL was purified from white asparagus spears successively by buffer extraction, ammonium sulfate precipitation, gel filtration (Sephacryl S-300), hydrophobic interaction chromatography (Phenyl-Sepharose), and fast protein liquid chromatography (FPLC). The purified PAL with 65-fold, and specific activity of 41 nkat/mg protein. Using Superose 12 column (FPLC), the molecular weight of asparagus PAL of native form was estimated to be 288 kD, the subunit weight was determined to be about 75 kD by SDS-PAGE, using immunoblot by bamboo PAL antibody, and the PAL might be a tetrameric form. Biochemical properties of asparagus PAL：The optimum temperature of PAL activity was 55.℃ The Km value for L-phenylalanine was 982 μM. The activation energy was 22.5 kcal/mol. The optimum pH of PAL activity was 8.5. Asparagus PAL activity is activated by Ca2+, Mg2+ and inhibited by Mn2+, Co2+, Hg2+. Secondary metabolites e.g. trans-cinnamic acid, p-amino-benzonic acid, caffeic acid and tannic acid with phenomena of feedback inhibition on asparagus PAL. PAL had specificity to L-phenylalanine. Enzyme catalytic site presented serine, tyrosine and histidine residues.