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研究生:施秋榮
研究生(外文):Chiu-Jung Shih
論文名稱:偏鄰苯二酚雙加氧酶的基理型抑制反應-3-胺基甲基兒茶酚的合成與反應機構探討
論文名稱(外文):Mechanism-Based Inactivation of Metapyrocatechase – Synthesis of 3-Aminomethylcatechol and Enyzmatic Reaction Mechanism
指導教授:黃得時黃得時引用關係
指導教授(外文):Ded-shih Huang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:化學系碩博士班
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:89
中文關鍵詞:基理型抑制偏鄰苯二酚雙加氧酶
外文關鍵詞:Metapyrocatechasemechanism-based inactivation
相關次數:
  • 被引用被引用:3
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  • 收藏至我的研究室書目清單書目收藏:1
3-胺基甲基兒茶酚可由2,3-二羥基苯甲醛與無水的氨縮合成3-亞胺基甲基兒茶酚,再經氫化而製備。
經由競爭型態分析後發現,3-胺基甲基兒茶酚與偏鄰苯二酚雙加氧酶的受質兒茶酚競爭同一個活性部位;且經偏鄰苯二酚雙加氧酶催化反應後掃瞄其波長300nm至600nm範圍內的吸收,發現在波長395 nm處有強烈的吸收,故推測其為偏鄰苯二酚雙加氧酶的受質(KM = 21.42 mM)。
當3-胺基甲基兒茶酚與偏鄰苯二酚雙加氧酶共同馴養時,酵素活性隨時間衰退,且該抑制反應為假一級反應,因此推測3-胺基甲基兒茶酚為偏鄰苯二酚雙加氧酶的基理型抑制劑,其KI = 39.85 mM,kinact = 1.29 min-1。
3-胺基甲基兒茶酚抑制偏鄰苯二酚雙加氧酶的反應機構仍須更進一步的光譜證明來說明。
The target substrate 3-aminomethylcatechol was prepared from imine derivative, which was in turn synthesized from the condensation of 2,3-dihydroxybenzaldehyde with ammonia.
3-aminomethylcatechol was proved to be a competitive inhibitor of metapyrocatechase. The enzyme mediated oxidation product was showed an intense absorption at 395 nm with a KM of 21.42 mM.
While incubated the metapyrocatechase with 3-aminomethylcatechol, the result revealed a a pseudo first-order time-dependent loss of enzyme activity, this result indicated that 3-aminomethylcatechol was a mechanism-based inactivatorof metapyrocatechase (KI = 39.85 mM,kinact = 1.29 min-1). A plausible inactivation mechanism was proposed.
目錄
中文摘要vi
英文摘要vii
圖目錄viii
表目錄xi

第一章 序論1
一、加氧酶1
二、芳香族開環斷裂雙加氧酶4
1 分類4
2 雙羥內、外雙加氧酶的催化反應比較6
3 鐵(II)雙羥外雙加氧酶的配位化學14
4 偏鄰苯二酚雙加氧酶的其他背景19
三、酵素動力學21
1 Michaelis-Menten 方程式21
2 競爭型抑制劑25
3 其他類型抑制之動力學28
(1) 非競爭型抑制28
(2) 未競爭型抑制29
四 基理型抑制31
1背景31
2 基理型抑制劑的判斷標準33
(1) 時間相關性33
(2) 飽和34
(3) 受質保護作用35
(4) 不可逆35
3 基理型抑制劑的主要應用方向36
(1) 酵素反應機構研究36
(2) 藥物設計36
4 偏鄰苯二酚雙加氧酶的基理型抑制劑37
五、研究動機37

第二章 實驗39
一、儀器與方法39
1 化學藥品39
2 二位天平39
3 四位天平39
4 核磁共振光譜儀39
5 旋轉濃縮儀39
6 氣相層析質譜儀39
7 烘箱39
8 無水溶劑系統40
9 真空系統40
10 酸鹼度計40
11 高溫高壓滅菌釜40
12 迴轉震盪培養箱40
13 無菌操作台41
14 超低溫冷凍箱41
15 紫外光譜儀41
16 加熱循環槽41
17 透析42
18 DEAE-cellulose離子交換樹脂前處理42
19 超過濾濃縮43
20 蛋白質分析43
21 十二磺酸平面膠體電泳44
22、電泳動力源48
23、高壓均質機48
24 高速冷凍離心機48
25 氫化器48
二、細菌培養48
三、偏鄰苯二酚雙加氧酶的純化51
四、基理型抑制劑3-胺基甲基兒茶酚的製備54
1 3-亞胺基甲基兒茶酚2的合成54
2 3-胺基甲基兒茶酚3的合成54
五、偏鄰苯二酚雙加氧酶的動力學研究55
1 兒茶酚與其衍生物的氧化反應55
2 受質的KM測試56
3 3-胺基甲基兒茶酚的抑制型態分析57
4 3-胺基甲基兒茶酚的基理型抑制反應57

第三章 結果 59
一、細菌培養59
二、酵素活性分析59
1 蛋白質濃度測定59
2 酵素純化活性分析60
3 電泳分析61
二、3-甲基胺基兒茶酚3的合成62
三、偏鄰苯二酚雙加氧酶的動力學研究分析63
1 兒茶酚的加氧反應63
2 受質的KM66
3 3-胺基甲基兒茶酚的抑制型態分析69
4 3-胺基甲基兒茶酚的基理型抑制反應73

第四章 討論 78
一、3-胺基甲基兒茶酚的合成78
二、偏鄰苯二酚雙加氧酶的動力學研究79
1 酵素活性79
2 基理型抑制反應80
3 反應機制的探討81

第五章 參考文獻 83
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