筊白筍苯丙胺酸裂解酶之生化學研究

Biochemical Studies of Phenylalanine Ammonia-Lyase from Water Bamboo (Zizania latifolia)

鄭仲淵(Zhong-Yuan Zheng)

指導教授 ： 李平篤

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.00607

摘要

苯丙胺酸裂解 (phenylalanine ammonia-lyase, PAL EC 4.3.1.5) 是植物 phenylpropanoids 生合成之第一個酵素。此酵素催化苯丙胺酸 (phenylalanine) 經過非氧化型脫氨反應產生肉桂酸 (trans-cinnamic acid)。這個反應導致植物二級代謝產物及大量的 phenylpropanoid 衍生物生合成，包括木質素 (lignin)、黃酮素 (flavonoid) 等。
本論文以筊白筍為材料；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，得到純化倍率為 110.5 倍，比活性為 53.3 nkat/mg protein。以膠體過濾法 (Superose 6, Superose 12) 測得之 PAL 原態分子量介於 290-300 kD，其次單元體分子量由 SDS-PAGE 測得約為 75 kD，因此推測筊白筍 PAL 可能為同質四元體結構。藉綠竹之 PAL 抗體進行西方點墨法顯示純化所得確實為 PAL，並將所得之 PAL 進行相關生化學研究
筊白筍與其他禾本科植物之 PAL 具有類似生化性質。苯丙胺酸裂解酶對基質 Phe 之 Km 值為筊白筍 516 μM。酵素最適反應溫度為 45℃。活化能為16.1 kcal/mol；最適反應 pH 為 8.5。鈣、鎂、錳在低濃度事會活化 PAL 活性；鈷、汞離子則會抑制。二級代謝產物，包括 trans-cinnamic acid、p-coumaric acid、ferulic acid、p-amino-benzonic acid 以及 caffeic acid，有產物回饋抑制現象。PAL 對基質 L-Phe 有專一性。酵素催化作用之活化中心具有 serine、tyrosine、histidine 等胺酸。

關鍵字

苯丙胺酸裂解酶

並列摘要

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is the first enzyme of the phenylpropanoid biosynthetic pathway. The enzyme catalyzes the non-oxidative deamination of phenylalanine to trans-cinnamic acid. This reaction leads to bio- synthesis of many phenylpropanoid-derived secondary metabolic products in plants, such as flavonoids and lignin.
In this study, PAL was purified from water bamboo by buffer extraction, ammonium sulfate precipitation, Sephacryl S-300, Phenyl- Sepharose, and Fast Protein Liquid Chromatography (FPLC). The purified PAL with 110.5-fold purification and specific activity of 53.3 nkat/mg protein obtained from water bamboo. Using Superose 6 and Superose 12 column (FPLC), the molecular weight of native form PAL was estimated to be 290-300 kD, the molecular weight of subunit form was determined to be 75 kD by SDS-PAGE.Using Western blot by antibody of bamboo PAL, and the results indicated that after the purification procedure the enzyme PAL had been purified. The pure enzyme was used to test biochemical properties.
PAL from water bamboo is similar with Gramineae plants in biochemical properties. The Km values for L-Phe were 516.The optimum temperature and pH were 45℃ and 8.5, respectively. The activation energy was 16.1 kcal/mol. Ca2+, Mg2+, Mn2+ actived PAL activity in low concentration; Co2+, Hg2+ inhibited PAL activity. Secondary metabolites including trans-cinnamic acid, p-coumaric acid, ferulic acid, p-amino-benzonic acid, and caffeic acid with phenomena of feedback inhibition. PAL has high substrate specificity to L-phenylalanine, and the presence of seryl, tyrosyl and histidyl groups was found to be essential for enzyme catalysis.

並列關鍵字

Phenylalanine Ammonia-Lyase

參考文獻

Alibert, G., Ranjeva, R. and Boudet, A. (1972) Studies on enzymes catalyzing phenolic acids formation in Quercus penduculata (Ehrh.). II. Intracellular location of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase and "benzoate synthase". Biochim Biophys Acta 279, 282-288.

Allwood, E.G., Davies, D.R., Gerrish, C., Ellis, B.E. and Bolwell, G.P. (1999) Phosphorylation of phenylalanine ammonia-lyase: evidence for a novel protein kinase and identification of the phosphorylated residue. FEBS Lett. 457, 47-52.

Alunni, S., Cipiciani, A., Fioroni, G. and Ottavi, L. (2003) Mechanisms of inhibition of phenylalanine ammonia-lyase by phenol inhibitors and phenol/glycine synergistic inhibitors. Arch Biochem Biophys. 412, 170-5.

Appert, C., Logemann, E., Hahlbrock, k., Schmid, J. and Amrhein, N. (1994) Structural and catalytic properties of the four phenylalanine ammonia-lyase isozymes from parsley (Petroselinum crispum Nym.). Eur J Biochem 225, 491-499.

Appert, C., Zon, J. and Amrhein, N. (2003) Kinetic analysis of the inhibition of phenylalanine ammonia-lyase by 2-aminoindan-2-phosphonic acid and other phenylalanine analogues. Phytochemistry. 62, 415-22.