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研究生:簡子烈
研究生(外文):Chane, Tze-Lieh
論文名稱:磷酸葡萄糖異構酉每中保留性組氨酸之功能分析
論文名稱(外文):The Function ofo the Conserved Histidine of Phosphoglucose Isomerase from Bacillus stearothermophilus
指導教授:孟孟孝
指導教授(外文):Meng, Meng-Hsiao
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農業生物科技學研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:中文
論文頁數:54
中文關鍵詞:組氨酸丙氨酸
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  磷酸葡萄糖異構(phosphoglucose isomerase, EC 5. 3. 1. 9)可促進六磷酸葡萄糖與六磷酸果糖中一號碳(C1)與二號碳(C2)間氫(hydrogen)的轉移,因此可催化六磷酸葡萄糖與六磷酸果糖的相互轉換,即異構作用。而氫的轉移被認為是由一個鹼基(bese)所起始(Dyson & Noltmann, 1968; Rose & O connell, 1961; Schray et al., 1973; Malaissie et al, 1990)。在各种不同來源的磷酸葡萄糖異構中發現有保留性的組氨酸(histidine)存在,被認為可能扮演異構作用中鹼基的角色。在本次研究中使用了定點突變的方法,以評估來自於Bacillus stearothermophilus的磷酸葡萄糖異構的保留性組氨酸,His311,是否可能扮演鹼基的角色。His31, His32 ,及His 311被個別以丙氨酸(alanine)取代之,由酵素動力學研究的結果顯示H311A的催化活性降低至只有野生型酵素活性的0.04%,而H31A及H32A的活性則沒有太大改變。再進一步將His311改變成天門冬醯胺(asparagine)及麩醯胺(glutamine),結果發現這兩個突變型酵素的催化效率,即Kcat/Km,與H311A的催化效相同。以上的結果暗示His311的功能可能是作為鹼基而不是氫鍵的提供或接受者。一種活化中心衍生型抑制劑(active-site directed inhibitor),N-Bromoacetylethanolamine phosphate,可以不可逆地抑制野生型磷酸葡萄糖異構的活性,然而H311A卻不受N-Bromoacetylethanolamine phosphate所抑制,由此可證實,由此可證實His311的確位於酵素的活化中心。由酵素之Kcat隨pH值變化的結果也支持His311在催化作用中乃是作為一個鹼基。
  Phosphoglucose isomerase (EC5.3.1.9) catalyzes the inter-conversion between D-glucopyranose 6-phosphate and D-fructofuranose 6-phosphate by promoting an intra-hydrogen transfer between C1 and C2 A base has been proposed to initate the transfer of hydrogen (Dyson & Noltmann, 1968; Rose & O'' connell, 1961; Schray et al, 1973; Malaisse et al 1990). A conserved histidine has been found throughout all phosphoglucose isomerases, and proposed to be the base catalyzing the isomerization reaction. In this study the conserved histidine, His311, of the enzyme from Bacillus stearothermophilus was studied by sitedirected mutagenesis to evaluate its possibility of being a base. His31, His32, and His311 were substituted with alanine individually. The kinetic analysis showed that the activity of H311A decreased to 0.04% of that of wild-type enzyme, while H31A and H32A remained unchanged in their activites His311 was further changed to asparagine and glutamine. The catalytic efficiency, Kcat/Km2 of these two mutant enzymes were similar to that of H311A, suggesting that His311 functions as a base instead of a hydrogen-bond donor or acceptor. N-bromoacetylethanolamine phosphate, an active-site directed inhibitor, inhibits irreversibly the activity of wild-type phosphoglucose isomerase. However, H311A became resistant to the inhibiton of N-bromoacetylethanolamine phosphate indicationg that His311 is really located in the active site of the enzyme. The result of the pH dependence of Kcat for the enzyme also suggests that His311 functioned as a general base in the catalysis.
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