耐輻射奇異球菌中歧鏈胺基酸轉胺酶與磷酸吡哆醛複合物之晶體結構分析

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研究生：	黃天鋒 Tien-Feng Huang
論文名稱：	耐輻射奇異球菌中歧鏈胺基酸轉胺酶與磷酸吡哆醛複合物之晶體結構分析 Crystal structure of branched-chain aminotransferase from Deinococcus radiodurans
指導教授：	陳俊榮 Chun-Jung Chen
口試委員:
學位類別：	碩士 Master
系所名稱：	理學院 - 物理學系 Department of Physics
論文出版年：	2006
畢業學年度：	94
語文別：	中文
論文頁數：	89
中文關鍵詞：	歧鏈胺基酸轉胺酶
相關次數：	點閱：2 下載：0
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歧鏈胺基酸轉胺酶(Branched-Chain Amino Acid Aminotransferase ; BCAT)其功能為催化歧鏈胺基酸：白胺酸(leucine)、異白胺酸(isoleucine)和纈胺酸(valine)的轉胺作用。
抗輻射奇異球菌(Deinococcus radiodurans)歧鏈胺基酸轉胺酶(DrBCAT)基因被選殖和表現在大腸桿菌中。抗輻射奇異球菌為嗜極微生物(Extremophile)，可以生長在游離輻射的環境，為了適應環境，其酵素應該會有比較特別的活性或穩定性。
本論文主要是利用X-ray繞射來解DrBCAT的3D晶體結構，在與其他已知的BCAT結構做比較。DrBCAT在非對稱性單元為一個二元體(dimmer)，每一個單元體可以區分為N區域與C區域，這兩個區域分別包含3個α螺旋和6個β摺疊，而輔酶PLP是落在兩個區域之間。
利用DrBCAT的結構和不同物種BCAT的結構比較，探討其熱穩定性和受質反應速率的差異。(一)對於熱穩定性：已經知道Thermus thermophilus HB8 (HB8 BCAT)和大腸桿菌(eBCAT)的歧鏈胺基酸轉胺酶比DrBCAT穩定，從整體結構可以發現HB8 BCAT和eBCAT是屬於三元體分子堆疊形式，鍵結的氫鍵數分別為12對和6對，經過晶格對稱，可以看成為六元體分子堆疊形式，此六元體中包含和DrBCAT相似的二元體分子堆疊，鍵結的氫鍵數分為為10對和13對，故以單元體來看，形成六元體的分子堆疊會比形成二元體堆疊穩定，因為多了三元體氫鍵的鍵結。(二)受質反應速率的快慢：從結構的活性區域附近可以看出兩個明顯的不同，一個是主要反應位置Lysine202與輔酶PLP的距離約3Å，無法形成共價鍵結，是遠大於其他結構可形成共價鍵距離約1.37 Å。DrBCAT因為無法形成共價鍵的關係，這樣可能使受質可以直接與輔酶PLP反應，而不需打斷共價鍵後再反應，因此造成反應速率加快 ; 另一個是在受質附近的一個不穩定的Loop(173-179)上的一個相對胺基酸Tyrosine，它的位置正好是在受質反應區的開口處，可能也會直接影響到反應速率。

目錄
第一章 序論
1.1耐輻射奇異球菌........................................................................1
1.2轉胺酶
1.2.1轉胺酶的分類.....................................................................3
1.2.2轉氨酶反應機制………………………………………….5
1.3 歧鏈胺基酸轉胺酶…………………………………………...8
1.4歧鏈胺基酸轉胺酶的應用與其研究目的…………………...11
第二章 材料和方法
2.1 長晶實驗
2.1.1 長晶方法………………………………………………...13
2.1.2 較佳長晶條件…………………………………………...14
2.1.3 長晶步驟………………………………………………...14
2.2 數據收集與處理……………………………………………..16
2.3 巨分子決定的原理與方法
2.3.1 X-Ray簡介………………………………………………19
2.3.2 結晶基本原理與蛋白質晶體…………………………...20
2.3.3 繞射圖(diffraction pattern)的取得………………………21
2.3.4 相位角(phase angle)的決定……………………………..23
2.3.5 分子置換法(Molecular Replacement Method)…………..24
2.4 DrBCAT結構的決定與精調…………………………………25
第三章 結果與討論
3.1 DrBCAT的分子結構………………………………………….33
3.2 DrBCAT二元體間的作用力………………………………….34
3.3 DrBCAT活性區域的猜測……………………………………35
3.4 其它種類的歧鏈胺基酸轉胺酶晶體堆疊形式比較………...37
3.5 分子堆疊形式與熱穩定性關係的推測……………………...37
3.6 活性區域附近胺基酸與Specific activity關係推測…………38

參考資料………………………………………………………………..82

圖目錄

Fig.1  (A) PLP的結構式，(B) 三個疏水性(hydrophobic)的歧鏈胺基酸………………………...……………………………………..41
Fig.2  Reaction mechanisms of aminotransferase………….………….42
Fig.3  Stereospecificity for the hydrogen transfer….………………….43
Fig.4 氣體擴散結晶方法………………………………………………44
Fig.5  DrBCAT晶體……..…………………………………………….45
Fig.6 分子取代法基本圖像原理………………………………………46
Fig.7  hBCATc與DrBCAT比對相似程度…...……………………….47
Fig.8  Ramachandran Plot….………………………………………….48
Fig.9  Amino acid alignment .…………………………………………49
Fig.10  DrBCAT單元體分子結構…………………………………….50
Fig.11  (A) DrBCAT二元體結構，(B) A、B chain 比較……………..51
Fig.12  A-chain和B-chain個別胺基酸的RMSD値………………....52
Fig.13  A-chain和B-chain的B-factor大小….……………………….53
Fig.14 輔酶PLP與附近的胺基酸的鍵結……………………………..54
Fig.15 輔酶PLP附近胺基酸相對位置………………………………..55
Fig.16 五種不同物種的歧鏈胺基酸轉胺酶Lysine和輔酶PLP的距離
                                 ………………………………56
Fig.17 大腸桿菌歧鏈胺基酸轉胺酶(eBCAT)受質-Glutamate和附近胺基酸與DrBCAT相關胺基酸的疊印…………………………….57
Fig.18 人類粒線體歧鏈胺基酸轉胺酶(hBCATm)受質-Isoleucine和附近胺基酸與DrBCAT相關胺基酸的疊印…………………….58
Fig.19 猜測DrBCAT的受質反應位置………………………………..59
Fig.20 受質反應區與輔酶PLP相關鍵結……………………………..60
Fig.21  HB8 BCAT與DrBCAT二元體推疊形式.……………………61
Fig.22  eBCAT與DrBCAT二元體推疊形式..……………………….62
Fig.23 五個不同物種單元體的比較…………………………………..63
Fig.24  DrBCAT和eBCAT在受質接受區和Try胺基酸的相對位置.64
Fig.25 活性位置所帶的電荷分佈……………………………………..65

表目錄

Table  1  Folds of PLP-dependent enzymes………………………….66
Table 2 Families of PLP-dependent enzymes having reported three-dimensional structure………………………………...69
Table  3  Classification of aminotransferase………………………….70
Table  4  Amino donor specificity of BCATs………………………...71
Table  5  DrBCAT的晶體繞射和結構決定資料統計………………72
Table  6  表(A)為在日本收集的繞射數據統計，內含有每一層解析度 R Sym値 ; 表(B)為在台灣收集的繞射數據統計…………73
Table  7  表(A)與表(B)分別表示在日本與台灣收集的繞射數據的重複性(Redundancy)………………………………………….74
Table  8  DrBCAT結構表面積計算…………………………………76
Table  9  DrBCAT兩單元體間的胺基酸……………………………75
Table  10  A、B chain間由main-chain原子與其它原子所造成的氫鍵…………………………………………………………..76
Table  11  A、B chain間由side-chain原子與其它原子小於4 Å距離的組合，共58組。………………………………………77
Table  12  輔酶PLP與附近胺基酸的距離………………………….78
Table  13  輔酶PLP與猜測的受質反應區胺基酸的距離………….79
Table  14  表(A)是大腸桿菌歧鏈胺基酸轉胺酶三元體間main-chain的氫鍵，表(B)是HB8 歧鏈胺基酸轉胺酶三元體間main-chain的氫鍵………………………………………...80
Table  15  表(A)是大腸桿菌歧鏈胺基酸轉胺酶三元體間main-chain的氫鍵，表(B)是HB8 歧鏈胺基酸轉胺酶三元體間main-chain的氫鍵…………………………..81

                                

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