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) ，為一雙體結構，其功能在催化6-磷酸葡萄糖 (6-phospho-glucose, 簡稱G6P) 與6-磷酸果糖 (6-phosphofructose, 簡稱F6P) 間可逆性的轉換。PGI在醣解作用 (glycolysis)、醣質新生作用 (gluconeogenesis) 及五碳醣循環 (pentose phosphate cycle) 扮演著重要的角色，因此當人體6-磷酸葡萄糖異構酵素基因突變時，會造成遺傳性非球型溶血性貧血 (Hereditary nonspherocytic haemolytic anaemia) ，如今6-磷酸葡萄糖異構酵素缺乏已成為紅血球酵素缺乏導致遺傳性貧血病因的第三位。為了探討6-磷酸葡萄糖異構酵素先天性遺傳突變在酵素結構上對活性及穩定性的影響，挑選PGI遺傳性突變胺基酸進行點突變，並送入大腸桿菌表現，純化蛋白質進行酵素活性及穩定性的分析。初步分析現有的突變酵素，R75G、R83W、V101M、T195I、S278L、R347H、R347C、R472H、L487F及I525T可測得催化活性。酵素催化F6P轉換成G6P的活性測試方面，發現除了S278L的酵素催化反應效率常數 (kcat/KM) 約為野生株酵素 (wild type, 簡稱WT) 的0.1 %外，其餘突變株酵素約為WT的3到27 %。而酵素催化G6P轉換成F6P的活性測試方面，除了S278L於4 mM F6P反應速率約為WT的0.03 %外，其餘突變株酵素約為WT的2到37 %。在熱穩定方面，分別測試突變株酵素於50 ℃以及50 ℃含10 μM F6P下的半致死期 (half-life, 簡稱t1/2) ，結果顯示除了I525T的t1/2與WT差異不大外，其餘突變株酵素的t1/2皆不到WT的一半。另外，R273H、A300P、L339P、T375R、H389R及E495K則偵測不到活性。

Phosphoglucose isomerase (EC 5.3.1.9, PGI) is a dimeric enzyme that catalyzes the reversible interconversion of glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) . PGI plays an important role in glycolysis, gluconeogenesis and pentose phosphate cycle. 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 enzymes were generated by site-directed mutagenesis and expressed in E. coli. In the present study, the ten mutant enzymes including R75G, R83W, V101M, T195I, S278L, R347H, R347C, R472H, L487F and I525T are active. The specificity constant (kcat/KM) of converting F6P to G6P of the mutant enzyme S278L remained approximately 0.1 %, and the other active mutants remained 3 to 27 % activity compared with the wild type (WT). The rate velocity (in 4 mM F6P) of converting G6P to F6P of mutant enzyme S278L remained approximately 0.03 %, the other active mutants remained about 2 to 37 % activity compared with the WT. Regarding thermostability, the half-lives (t1/2) of various enzymes at 50 ℃ with or without the presence of 10 μM F6P, were determined, and the results showed that the t1/2 of mutant enzymes is shorten more than half compared with the WT except I525T. The other enzymes, R273H, A300P, L339P, T375R, H389R and E495K are inactive.

中文摘要 I
英文摘要 II
目錄 III
表目錄 VI
圖目錄 VII
第一章、前言 1
第一節、6-磷酸葡萄糖異構酵素之特性 1
第二節、6-磷酸葡萄糖異構酵素之催化機制假說 3
第三節、研究動機與目的 8
第二章、材料與方法 11
第一節、材料介紹 11
第二節、酵素的大量製備及純化 11
第三節、蛋白質定量 14
第四節、蛋白質電泳分析 14
第五節、蛋白質西方墨點法分析 15
第六節、人類6-磷酸葡萄糖異構酵素之酵素動力分析
(kinetic assay) 16
第七節、酮糖化學呈色法 20
第八節、人類6-磷酸葡萄糖異構酵素之熱穩定分析 21
第三章、結果 22
第一節、人類6-磷酸葡萄糖異構酵素的表達與純化 22
第二節、人類6-磷酸葡萄糖異構酵素催化6-磷酸果糖
轉換成6-磷酸葡萄糖的酵素動力學分析 22
第三節、人類6-磷酸葡萄糖異構酵素催化6-磷酸葡萄糖
轉換成6-磷酸果糖的酵素動力學分析 23
第四節、人類6-磷酸葡萄糖異構酵素的活性熱穩定分析 24
第五節、無活性之6-磷酸葡萄糖異構酵素突變株蛋白質
的表現狀況 25
第四章、討論 26
第一節、病人(未命名)之同源性基因突變(R83W/R83W) 26
第二節、病人(MM)之同源性基因突變(V101M/V101M) 27
第三節、病人(UC)之同源性基因突變(R472H/R472H) 27
第四節、病人(GPI-KS)之同源性基因突變(I525T/I525T) 28
第五節、病人(Homburg)之異源性基因突變(H20P/L339P) 29
第六節、病人(未命名)之異源性基因突變(T224M/E495K) 29
第七節、病人(未命名)之異源性基因突變(R273H/R347C) 30
第八節、病人(未命名)之異源性基因突變(S278L/L487F) 31
第九節、病人(JG)之異源性基因突變(A300P/R347C) 32
第十節、病人(Kinki)之異源性基因突變(T375R/D539N) 32
第十一節、病人(Calden)之異源性基因突變(Ｈ389R/L517V) 33
第十二節、病人(Elyria)之異源性基因突變(R75G/R96stop) 34
第十三節、病人(Bari)之異源性基因突變(R96stop/T195I) 34
參考資料 36
表 44
圖 52

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