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研究生:徐瑤蓉
研究生(外文):Yau-Jung Hsu
論文名稱:以化學救援法探究鴿肝蘋果酸第162位置離胺酸殘基在催化過程中所扮演的角色
論文名稱(外文):Studies on the Functional Role of Lysine 162 Residue in Pigeon Liver Malic Enzyme by Chemical Rescue
指導教授:周慰遠
指導教授(外文):Wei-Yuan Chou
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
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
中文關鍵詞:化學救援法蘋果酸催化
外文關鍵詞:Chemical RescueMalic EnzymeCatalysis
相關次數:
  • 被引用被引用:1
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  • 下載下載:15
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鴿肝蘋果酸 (E.C. 1.1.1.40) 能催化可逆性氧化脫羧反應,將蘋果酸分解為丙酮酸及二氧化碳,並使NADP+還原為NADPH。此在催化過程上為一普遍性酸鹼催化機制 (general acid-base mechanism),且需要二價金屬離子 (錳或鎂) 參與。鴿肝細胞漿蘋果酸之X-ray 晶體結構顯示分子上第162位置離胺酸 (Lys162) 可能和催化有關。在過去的研究亦說明Lys162在催化上具有決定性之影響。本論文以化學救援法 (chemical rescue) 探究鴿肝蘋果酸殘基Lys162於催化上所扮演之角色,並嘗試回復突變型K162A之活性。結果顯示,突變型K162A於基本動力學上之kcat與KmMal分別為野生型之10-5與38倍,KmNADP則和野生型沒有差別。於丙酮酸半反應上,突變型K162A之kcat和野生型沒有差別,但草醯乙酸脫羧反應之kcat則比野生型小668倍。外加氯化胺 (ammonium chloride) 在不影響分子整體結構下,使突變型K162A之kcat值回復100倍,KmMal回復6.5倍。於脫羧反應之活性回復3.5倍,氧化還原反應則沒有活性回復之情形。因此,鴿肝蘋果酸殘基Lys162可能作為一催化酸參與催化反應,而外加胺類則可以使突變型K162A回復部份活性及催化能力,於基本動力學之特性也較接近野生型鴿肝蘋果酸。
Pigeon malic enzyme catalyzes the reversible oxidative decarboxylation of malate to pyruvate and CO2, with concomitant reduction of NADP+ to NADPH. The enzyme follows the general acid-base mechanism and requires divalent metal, Mn2+ or Mg2+. The crystal structure of pigeon malic enzyme shows that lysine 162 (Lys162) may be involved in the catalysis. The pH profile and mutation studies suggest that Lys162 may serve as a general acid in the catalytic mechanism. In this study, we use a chemical rescue method to examine the general acid role of Lys162. The kcat value for K162A is 105-fold lower than that of wild type, whereas the KmMal value is 38-fold larger than that of the wild type. There is no effect on the value of KmNADP. For pyruvate reduction reaction, the kcat value for mutant is identical to that of the wild type. The kcat value for the oxalacetate decarboxylative reaction is reduced 668-fold for K162A. In the chemical rescue study, addition of ammonium chloride (100 mM) exhibited a ~100-fold increase in kcat and ~6.5-fold decrease in KmMal, respectively. The activity of the decarboxylation is partially restored by 3.5-fold. Therefore, the exogenous amine can be regard as a functional group representing Lys162 of the wild type enzyme. Lys162 probably acts as the general acid catalyst in the pigeon malic enzyme.
頁次
縮寫表…………………………………………………………… II
表目錄……………… …………………………………………… III
圖目錄………………………………………………………… IV
中文摘要…………………………………………………… V
英文摘要……………………………………………………… VI
壹、緒論……………………………………………………… 1
貳、實驗材料與方法
一、藥品試劑……………………………………………………… 6
二、緩衝液及培養液之製備…………………………………… 7
三、主要儀器與器材……………………………………………… 9
四、實驗方法
(一) 重組突變型K340A鴿肝蘋果酸 ( pET-15b-ME (K340A))
製備………………………………………………… 9
(二) 重組野生型及各突變型鴿肝蘋果酸之純化………… 12
(三) 自然電泳與染活性分析………………………………… 16
(四) 重組鴿肝蘋果酸活性分析…………………………… 17
(五) 重組鴿肝蘋果酸結構分析…………………………… 21
參、實驗結果………………………………………………… 23
肆、討論……………………………………………………… 32
表……………………………………………………………… 40
圖……………………………………………………………… 45
參考文獻……………………………………………………… 64
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