臺灣博碩士論文加值系統

6-磷酸葡萄糖異構酵素(phosphoglucose isomerase，E.C. 5.3.1.9；簡稱PGI)，在生理上主要參與醣類代謝中的醣解作用（glycolysis）及醣質新生作用（gluconeogenesis）中6-磷酸葡萄糖（6-phosphoglucose，G6P）與6-磷酸果糖（6-phosphofructose，F6P）間的轉換，由於6-磷酸葡萄糖和6-磷酸果糖在醣解作用、醣質新生作用及五碳醣磷酸循環（pentose phosphate cycle）扮演極為重要的角色，因此當人體6-磷酸葡萄糖異構酵素因突變而發生缺損時，會造成遺傳性溶血性貧血（Hereditary nonspherocytic haemolytic anaemia）。為了探討先天遺傳突變對6-磷酸葡萄糖異構酵素結構、活性及穩定性的影響，我們將人體6-磷酸葡萄糖異構酵素的基因選殖入pUC18，並利用點突變法（Site-direct mutagenesis）構築具有先天性突變的6-磷酸葡萄糖異構酵素基因，然後將先天性突變的6-磷酸葡萄糖異構酵素基因送入大腸桿菌表現，再利用陰離子交換層析管柱及陽離子交換層析管柱純化蛋白，最後進行酵素活性及穩定性的分析。各突變酵素對受質F6P的酵素催化反應速率常數（kcat/KM）分別如下：R83W（Arg83突變為 Trp之變異酵素，以下之突變以此原則命名）、V101M、R347C及R472H約為wild type（WT）的50 ﹪，R347H 約為WT的30 ﹪，而I524T約為WT的5 ﹪。另外R273H即使在分析時加入大量酵素下也無法測得活性，而S278L及E495K則無法以同樣純化方法獲得蛋白，而且在生產此兩蛋白之細胞萃取液中也無法測得活性。在熱穩定方面，我除了測量各個突變酵素在50℃的熱穩定性外，還測量各個突變酵素在過量的F6P（1 mM）存在下及正常生理濃度的F6P (10 μM)下的半衰期（t1/2）。結果顯示除了I524T的熱穩定性與WT差異不大外，其餘的突變蛋白即使在加入1 mM 的F6P下，熱穩定性也遠低於WT。配合已知的人類6-磷酸葡萄糖異構酵素之立體結構，本篇論文也嘗試討論各個突變點影響蛋白穩定性的原因。未來希望繼續完成其他突變蛋白的分析。在各個突變點造成結構上的推測，我也想用更多的實驗證據來證明我的論點。

. Phosphoglucose isomerase （E C 5.3.1.9;PGI） is a dimeric enzyme that catalyzes the reversible inter-conversion of glucose-6-phosphate（G-6-P）and fructose-6-phosphate（F-6-P） in glycolysis and gluconeogenesis pathways. Phosphoglucose isomerase deficiency is the third most common enzyme deficiency known to cause hereditary nonspherocytic hemolytic anemia. Our aim is to analyze the catalytic efficiency and structural stability of the inherited mutant phosphoglucose isomerase. The mutated proteins were generated by site-directed mutagenesis and expressed in E. coli. The activity assays show that mutant proteins R83W、V101M、R347C and R472H decrease approximately 2 folds, R347H decreases approximately 3 folds, and I524T decreases approximately 20-fold compare with the kcat/KM of wide type enzyme. R273H has no activity even a large amount of the enzyme was used for activity assay. S278L and E495K could not be obtained under the same purification conditions suggesting that they have problem in protein folding. Regarding thermostability, the half-lives (t1/2) of mutated proteins were much less stable than WT event at saturated concentration of substrate. I524T is slightly unstable compared with wild-type enzyme at 55 ℃ with 1 mM F-6-P. Combined with the 3-D structure of human phosphoglucose isomerase, I also attempt to address the reason causing the decrease of thermostability of each mutant PGI protein. More hereditary mutated PGI proteins will be characterized in the future to address the structure-function relationship of the hereditary mutations of phosphoglucose isomerase.

目錄 頁
中文摘要 I
英文摘要 III
目錄 IV
圖目錄 VI
表目錄 IX
壹、 前言 1
6-磷酸葡萄糖異構酵素之功能 1
6-磷酸葡萄糖異構酵素之X光結晶學研究 4
目前對6-磷酸葡萄糖異構酵素提出的催化機制 8
研究動機與目的 11
貳、 材料與方法 14
質體之構築介紹 14
菌株、質體及培養基 14
聚合酶鏈鎖反應定點突變法 14
DNA 黏接反應 16
質體轉型作用 16
核苷酸定序法鑑定突變株 17
酵素大量製備及純化 18
蛋白質電泳分析 20
蛋白質定量 21
6-磷酸葡萄糖異構酵素之氨基酸定序比對分析 22
酮醣定性分析 23
酵素動力學分析 25
參、結果 30
人類6-磷酸葡萄糖異構酵素基因的蛋白表現 30
人類6-磷酸葡萄糖異構酵素的純化與比對 31
催化6-磷酸果醣轉換成6-磷酸葡萄糖的酵素動力學分析 32
催化6-磷酸葡萄糖轉換成6-磷酸果醣的酵素動力學分析 33
6-磷酸葡萄糖異構酵素的熱穩定分析 34
肆、討論 35
伍、未來展望 41
陸、圖和表 42
柒、參考文獻　　　　　　　　　　　　　　　　　　 　 　74

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