臺灣博碩士論文加值系統

酪胺酸酶 (Tyrosinase, EC 1.14.18.1) 為含銅的氧化酶，在黑色素 (Melanin) 生合成途徑中，為一個重要的酵素，酪胺酸酶催化反應主要將酪胺酸 (Tyrosine) 羥基化，轉變成多巴 (L-DOPA)，進一步將多巴氧化成多巴醌 (Dopaquinone)，最後生成黑色素，在哺乳動物中，黑色素決定了毛髮及皮膚的顏色。
本研究主要建立酪胺酸酶之酵素動力學，以酪胺酸為受質探討熊果素 (Arbutin)抑制蘑菇酪胺酸酶的酵素動力學，並證明熊果素為競爭型抑制劑，以確認系統的可靠信。此外，在 2006 年已經解出鏈黴菌 (Streptomyces-Castaneoglobisporus) 酪胺酸酶的晶體結構，並以此結構為基礎，以同源模擬的方式，建立蘑菇及人類酪胺酸酶蛋白質結構，接著以分子對接的軟體探討熊果素和蘑菇及人類酪胺酸酶鍵結的形式，結果證明熊果素苯環上面的氫原子可和酪胺酸酶活化位中 peroxide 的氧離子產生氫鍵，達到抑制酪胺酸酶的活性，進一步以分子對接的方式證明，熊果素鍵結在活化位的位置為競爭型抑制劑。
另一方面以酵素活性測試的實驗來測試，由虛擬高速藥物篩選出來的九個化合物，是否具有抑制酪胺酸酶活性的能力，由實驗結果顯示：4-CPD 和 5-CPD 具有抑制酪胺酸酶活性的能力，在濃度 0.8mM下，抑制能力為5-CPD > 4-CPD > arbutin。由酪胺酸酶活性測試篩選結果，得知 1-CPD~9-CPD 當中，以 5-CPD 具有最佳抑制酪胺酸酶活性的能力，接著尋找出九個 5-CPD 的相關衍生物，分別為 5.1-CPD、5.2-CPD、5.3-CPD、5.4-CPD、5.5-CPD、5.6-CPD、5.7-CPD、5.8-CPD、5.9-CPD 進行 in vitro 實驗，最後實驗結果顯示 5-CPD 有最好的抑制酪胺酸酶活性的效果。

Tyrosinase is a copper containing oxidase that catalyzes two distinct reactions of melanin synthesis: the hydroxylation of tyrosine to L-DOPA and the oxidation of L-DOPA to dopaquinone which then polymerize to form pigments. In animals, melanin is secreted by melanocyts that determine the color of skin and hair.
In our present study, we set up kinetics of mushroom tyrosinase in oxidation of L-tyrosine. The effect of arbutin on the monophenolase activity of mushroom tyrosinese has been studied. The results show that arbutin inhibit monophenolase activity of mushroom tyrosinase and acted as a competitive inhibitor. In 2006, the crystal structure of tyrosinase from Streptomyces-Castaneoglobisporus was solved and structure-based catalytic mechanism of tyrosinase was understood. Base on the crystal structure of Streptomyces-Castaneoglobisporus tyrosinese, build structure models of mushroom and human tyrosinese by homology modeling. Arbutin exhibited competitive binding at the active site in virtual model of molecular docking of human tyrosinese.
On the other hand, we test inhibitory effects of nine compounds from virtual screening. The results show that 4-CPD and 5-CPD can inhibit the activity of mushroom tyrosinase. The rank order of inhibition was: 5-CPD > 4-CPD > arbutin. The result of tyrosinase activity screening showed that 5-CDP inhibited tyrosinase activity to be perfect between 1-CDP~9CDP. Then we find nine related derivatives of 5-CPD which among 5.1-CPD, 5.2-CPD, 5.3-CPD, 5.4-CPD, 5.5-CPD, 5.6-CPD, 5.7-CPD, 5.8-CPD and 5.9-CPD, respectively. The results show that 5.1-CPD, 5.2-CPD, 5.3-CPD, 5.4-CPD, 5.7-CPD, 5.8-CPD and 5.9-CPD can inhibit the activity of mushroom tyrosinase and the most inhibited tyrosinase activity is 5-CPD.

目錄
英文摘要 5
中文摘要 6

第一章 緒論
1-1 皮膚構造與黑色素的關係 7
1-1-1 皮膚的構造與功能 7
1-1-2 表皮層的構造 7
1-2 黑色素的重要性及生合成途徑 7
1-3 酪胺酸酶的形式及催化作用機制 8
1-4 美白成分之作用機轉 9
1-4-1 酪胺酸酶去活化 9
1-4-2 競爭性取代酪胺酸酵素受質 10
1-4-3 其他影響酪胺酸酶之物質 10
1-5 分子對接軟體介紹 10
1-5-1直接藥物設計 10
1-5-2 GOLD 11
1-5-3 Surflex-Dock 12
1-5-4 Glide 14
1-6 研究動機與目的 15
第二章 材料與方法
2-1 In vitro 酪胺酸酶活性測試 17
2-1-1 藥品來源 17
2-1-2 藥品配製 17
2-2 In silico 分子對接 17
2-2-1 本研究中所使用的軟體 17
2-2-2 已知酪胺酸酶的蛋白質結構 18
2-2-3 建立 Mushroom 酪胺酸酶結構 18
2-2-4 建立人類酪胺酸酶結構 19
2-2-5 利用 PROCHECK 評估模擬結構的可靠性 19
2-2-6 分子對接的準備 19
第三章 結果與討論
3-1 In vitro酪胺酸酶活性實驗條件測試 21
3-2 In vitro酪胺酸酶活性測試結果 21
3-2-1 酵素動力學系統的建立 21
3-2-2 利用酪胺酸酶活性測試篩選結果 21
3-2-3 選出九個 5-CPD 衍生物進行酪胺酸酶活性測試 22
3-3 不同物種的酪胺酸酶蛋白質序列比對的結果及模擬的結構 22
3-3-1 Mushroom 酪胺酸酶結構 22
3-3-2人類酪胺酸酶結構 23
3-3-3 PROCHECK 評估模擬結構的結果 23
3-4 分子對接結果 23
3-4-1 以 Mushroom 酪胺酸酶結構分子對接的結果 23
3-4-2 以人類酪胺酸酶結構分子對接的結果 23
第四章 結論 25
第五章 參考文獻 26

圖表目錄
表一、以 Gold, Surflex, Glide 分子對接軟體分析，Mushroom酪胺酸酶和 Arbutin, kojic acid, 4-CPD, 5-CPD, 7-CPD, 8-CPD, 9-CPD 的分子對接結果 29
表二、以 Gold, Surflex, Glide 分子對接軟體分析，人類酪胺酸酶和 Arbutin, kojic acid, 4-CPD, 5-CPD, 7-CPD, 8-CPD, 9-CPD 的分子對接結果 29
表三、以 Gold, Surflex, Glide 分子對接軟體分析，Mushroom 酪胺酸酶和 5-CPD 及其衍生物的分子對接結果 30
表四、以 Gold, Surflex, Glide 分子對接軟體分析，人類酪胺酸酶和 5-CPD 及其衍生物的分子對接結果 30

圖一、不同濃度的DMSO對酪胺酸酶活性測試 31
圖二、不同濃度熊果素的吸光值 32
圖三、熊果素的Lineweaver–Burk plots 33
圖四、不同抑制劑濃度處理下，酪胺酸酶剩餘活性百分比 34
圖五、不同抑制劑濃度處理下，酪胺酸酶剩餘活性百分比 35
圖六、Mushroom 和鏈黴菌酪胺酸酶序列比對 36
圖七、人類和鏈黴菌酪胺酸酶序列比對 37
圖八、Mushroom 酪胺酸酶蛋白質結構 38
圖九、人類酪胺酸酶蛋白質結構 38
圖十、評估模擬 Mushroom 酪胺酸酶蛋白質結構的結果 39
圖十一、評估模擬人類酪胺酸酶蛋白質結構的結果 40
圖十二、熊果素 和 mushroom 酪胺酸酶分子對接的情形 41
圖十三、麴酸和 mushroom 酪胺酸酶分子對接的情形 42
附錄圖表
附錄圖一、表皮的構造 43
附錄圖二、表皮層中三種不同的細胞型態 44
附錄圖三、黑色素生合成途徑 45
附錄圖四、酪胺酸酶的形式及催化作用機制 46
附錄圖五、氧化態酪胺酸酶作用機制 47

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