Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23925
標題: I. 桿菌屬磷酸葡萄糖異構酵素中可能參與磷酸辨識結合之胺基酸殘基的功能分析。II. 人類及大腸桿菌磷酸葡萄糖異構酵素之大量表達系統及純化方法的建立。
I.The Functional Analysis of the Amino Acid Residues ofBacillus stearothermophilus Phosphoglucose Isomerasewhich May Recognize the Phosphate Group of Substrate. II.Establishment of the Overexpression and PurificationSystems for Phosphoglucose Isomerase fr
作者: 謝嘉榮
Hsieh, Chia-Jung
關鍵字: phosphoglucose isomerase
磷酸葡萄糖異構酵素
site-directed mutation
定點突變
出版社: 生物化學研究所
摘要: 磷酸葡萄糖異構酵素(phosphoglucose isomerase, E.C. 5.3.1.9)在糖解作用(glycolysis)及生糖作用(gluconeogenesis)中催化6-磷酸葡萄糖與6-磷酸果糖間的轉換,是極為重要的生理酵素。先前的研究報告指出Lys144→Ala時,基質之KM值變大,顯示在催化過程中Lys144可能與基質結合有關。為了進一步確定酵素活化中心中位於基質磷酸根周圍的胺基酸與磷酸根之相互關係,實驗上以Bacillus stearothermophilus 磷酸葡萄糖異構酵素B蛋白結構為基礎,建構基質與活化中心的結合模型。模型顯示:Lys144的側鏈可能直接與基質磷酸根有接觸。因此本研究工作主要以特定胺基酸(Lys144)在活性催化功能上進行動力學分析,更進一步確認它與基質上磷酸根辨識關係的重要性。另一方面磷酸葡萄糖異構酵素B 蛋白結構顯示G83、S84、G206在活化中心的空間架構上,均位於基質磷酸根的周圍,因此另外嘗試以定點突變的方法來改變空間架構,試著改變酵素與基質催化的特性,將原來的催化基質改變為葡萄糖,使已突變的磷酸葡萄糖異構酵素能夠催化葡萄糖轉換為果糖 ; 此外並配合動力學常數的分析,來確認各突變株的功能。由目前實驗研究結果指出,在K144、 G83、S84、G206的定點突變實驗中,對基質的催化活性均有降低的結果,顯示各突變位對催化活性有其重要性,但是仍無法改變酵素與基質的催化特性。 另外在先前研究報告指出,哺乳類磷酸葡萄糖異構酵素即是所謂的神經白細胞素,也是癌細胞自行分泌的autocrine motility factor。因此在本實驗室除了以B. stearothermophilus的磷酸葡萄糖異構酵素作為研究對象外,並嘗試選殖哺乳類(人)之磷酸葡萄糖異構酵素作研究,期望能建立蛋白大量表現及純化系統,以利各接續的實驗進行。由目前實驗結果得知其基因已被選殖出,但無法在大腸桿菌中大量表現。而我們也由已知的磷酸葡萄糖異構酵素胺基酸序列中,得知大腸桿菌與人類之磷酸葡萄糖異構酵素有70%的相似性 ; 因此在無法大量表現人類磷酸葡萄糖異構酵素時,是否可以以大腸桿菌磷酸葡萄糖異構酵素為研究對象,來推論並回答在人類磷酸葡萄糖異構酵素的遺傳疾病問題。所以在另一個實驗中:[大腸桿菌磷酸葡萄糖異構酵素的大量表現及純化系統的建立],嘗試對大腸桿菌磷酸葡萄糖異構酵素蛋白,進行大量表現及純化系統的建立,並對純化的蛋白作結晶工程實驗,與B. stearothermophilus 磷酸葡萄糖異構?A在結構上做分析比較,以瞭解耐熱性及不耐熱性的磷酸葡萄糖異構酵素結構上有何差別。在目前實驗中,其大量表達系統及純化系統已在實驗室中建立,且也得到相當純度的蛋白,並進一步對已純化的蛋白做結晶工程。
Phosphoglucose isomerase (E C 5.3.1.9) is a dimeric enzyme that catalyzes the reversible inter-conversion of glucose 6-phosphate and fructose 6-phosphate in glycolysis and gluconeogenesis. There are two isozymes of phosphoglucose isomerases in Bacillus stearothermophilus; namely Pgi A and Pgi B. Our experiment is based on the protein structure of Pgi B that points out G82, S83, K139 and G201 are all surrounding the phosphate group of substrate (SUN, et al, 1999). In our previous study, the substitution at position K144RA of Pgi A(corresponding to K139 of Pgi B) resulted in 5-fold increases in KM, and 3.7-fold decreases in kcat (MENG, et al,1998). Such increases agree with the suggestion that K144 is located at the active site. My primary aim is to analyze the function and determine the importance of G83, S84, K144 and G206 of Pgi A(corresponding to G82, S83, K139 and G201 of Pgi B) in recognizing phosphate group on the substrate. Site-directed mutation and kinetic analysis were used to analyze the enzyme catalytic characteristics. The activities of all mutant proteins were approximately 7~80 fold decreases in kcat/KM, suggesting that G83, S84, K144 and G206 play important roles in enzyme catalytic activity. The deficiency of phosphoglucose isomerase in human leads to nonspherocytic hemolytic disease (Beutler, E., et al, 1997). Recently, it has been shown that neurolukin(NLK), autocrine motility factor(AMF), and maturation factor(MF), are analogous to phosphoglucose isomerase. In order to study the function of the mammalian Pgi. We would like to establish the overexpression and purification systems for human Pgi. In this study, we were enable to overexpress human Pgi in Escherichia coli. The pgi gene of E. coli was also amplified from the human cDNA library contaminated with E. coli cells in an PCR reaction by the primers designed for the amplification of human pgi gene due to the high similarity of nucleotide sequences between those two pgi genes. The overexpression and purification systems of E. coli Pgi have been established in the lab.
URI: http://hdl.handle.net/11455/23925
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