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National Chung Hsing University Institutional Repository - NCHUIR > 農業暨自然資源學院 > 生物科技學研究所 > 依資料類型分類 > 碩博士論文 >  Bacillus stearothermophilus 6-磷酸葡萄糖異構酵素-A中活化中心之酸性氨基酸的功能分析

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/97786

標題: Bacillus stearothermophilus 6-磷酸葡萄糖異構酵素-A中活化中心之酸性氨基酸的功能分析
Function Analysis of the Conserved Anionic Residues of Bacillus stearothermophilus 6-Phosphoglucose Isomerase-A
作者: 林華洋
Lin, Huayang
Contributors: 孟孟孝
Menghsiao Meng
國立中興大學
關鍵字: 磷酸葡萄糖異構酵素;異構酵素;催化機制
Bacillus stearothermophilus;phosphoglocose isomerase;glucosephospho isomerase;pgi;gpi;ph profile;catalysis
日期: 2000
Issue Date: 2012-09-04 09:20:27 (UTC+8)
Publisher: 農業生物科技學研究所
摘要: 6-磷酸葡萄糖異構酵素(EC 5.3.1.9)是一種具有多種功能的酵素。除了參與醣酵解路徑,6-磷酸葡萄糖異構酵素在胞外並有當作促神經細胞生長因子,促腫瘤移動因子的能力。最近的研究更發現,6-磷酸葡萄糖異構酵素也是自體免疫性風濕性關節炎老鼠內T細胞及免疫球蛋白的結合目標。在本實驗中,我們將取自Bacillus stearothermophilus 的6-磷酸葡萄糖異構酵素,利用定點突變分析,對一些保留的陰電性氨基酸,Glu150, Glu290, Glu401,Glu422,和Asp417,將之替換為alanine (A),aspartic acid (D), asparagine (N),及 glutamine (Q)。在酵素動力學的分析實驗中D417A,E422A突變酵素的kcat/Km值,相較於野生株蛋白,分別降低了十倍和五倍,E401A突變酵素則近乎不變。而E150A及E150D、E150N,E150Q突變蛋白的kcat/Km變化,與野生株蛋白相較,則有一千倍、三倍、一萬及十三萬倍的降低。另外,在E290A,E290Q突變蛋白,兩者的kcat/Km 值都有約兩千倍的降低,而E290D,E290N突變蛋白的kcat/Km 值,則降低了七千倍及十二萬倍。這結果說明了,E150,E290,這兩個氨基酸殘基在催化過程中扮演了重要的角色。而在pH profile的實驗中,H311Q,E150A,及 E150N 突變蛋白,其pKa 1相較於野生株蛋白有下降的趨勢。 這些pKa 上的變化,與這些氨基酸在經突變後電性變化應有一定關聯。再配合已知的葡萄糖磷酸化異構酵素X光結晶結構圖,我們推測,在催化過程中,H311氨基酸殘基有正電荷的累積,E150 氨基酸殘基可能是當作一陰電性機團利用靜電引力 (electrostatic force)來穩定H311以降低活化能。而E290氨基酸殘基則是在催化過程中作為一general acid來參與催化過程。
Phosphoglucose isomerase (EC 5.3.1.9) is a multi-function protein that catalyzes the interconversion of D-glucopyranse-6-phosphate and D-fructofuranose-6-phosphate in the glycolysis and gluconeogenesis pathway. It is also identical to neuroleukin, autocrine motility factor (AMF) and is the target of both the initiating T cell and pathogenic immunoglobulins in arthritic mouse. In this study, we replaced the conserved glutamic acid (E150, E290, E401 and E422) and aspartic acid (D417) of phosphoglucose isomerase A from Bacillus stearothermophilus with alanine (A), aspartic acid (D), asparagine (N), or glutamine (Q) by site-directed mutagenesis. In general, the Km was varied insignificantly, while the change of kcat was varied in a great extent. The D417A and E422A mutants had 10-fold and 5-fold decrease in kcat/Km relative to the wide-type enzyme, respectively; whereas the E401A mutant had no significant change in kcat/Km relative to the wide-type enzyme. The E150A, E150D, E150N, and E150Q mutants had 1000-fold, 3-fold, 10000-fold and 130000-fold decrease in kcat/Km relative to the wide-type enzyme. Both E290A and E290Q mutants had 2000-fold decrease in kcat/Km relative to the wide-type enzyme. E290D and E290N mutants had 7000-fold and 120000-fold decrease in kcat/Km relative to the wide-type enzyme. Thus, the data suggest that the role of the E150 and E290 residues are different in the catalytic process. In our pH profil study, we show the significant pKa change in some mutants , E311Q, E150A, and E150N. The E311Q, E150A and E150N mutants had some decrease in pKa 1. We assume that the change of pKa relates to the different electric charge between the wild type amino acid residues and substitution. According to the data presented here and 3-D crystal structure of phosphoglucose isomerase-B, we propose that the E150 residue stabilizes the developing-positive charge in H311 that functions as a general base and as a result, reduces the activation energy by electrostatic force. The E290 residue plays a role as a general acid in isomerization step.
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