臺灣博碩士論文加值系統

鴿肝蘋果酸酶催化可逆性的氧化脫羧反應，將蘋果酸分解成丙酮酸及二氧化碳，並使NADP+還原為NADPH。反應過程需要二價金屬離子 (錳或鎂) 的參與，並依循一普遍性酸鹼催化機轉 (general acid-base mechanism) 進行作用。從鴿肝蘋果酸酶的結晶結構及和金屬催化氧化系統，認為D234、D235與D258應是鴿肝蘋果酸酶金屬結合的配位體，而在蛔蟲 (Ascaris suun) 蘋果酸酶的研究中指出，D295 (相當於鴿肝蘋果酸酶的D258) 在催化上被認為是作為一催化鹼的功能，因此在鴿肝蘋果酸酶中D258可能也是作為催化鹼。此外，從結晶結構及以前定位突變的研究結果推論K162可能是作為反應中的催化酸。
為了更進一步確定這兩個胺基酸在鴿肝蘋果酸酶催化反應上所扮演的角色，將K162和D258其分別突變為丙胺酸 (Alanine)，由初速實驗結果顯示，K162A和D258A的kcat值分別比野生型小26300及7700倍。K162A在被不同濃度的TNBS修飾後，在二十分鐘內活性並不隨時間改變。於丙酮酸半反應上，突變型K162A之kcat和野生型差不多，但草醯乙酸脫羧反應之kcat則比野生型小3262倍。同時K162A的半反應速率比 (rH) 的值比野生型小6.5倍; 而K162A的pH profile中顯示在高pH時K162A的kcat維持不變。因此我們認為K162的氨基 (ε-amino group) 扮演催化酸的角色，提供質子給烯醇式丙酮酸的三號碳使之轉變為酮式丙酮酸。
於D258A的丙酮酸還原半反應及草醯乙酸脫羧半反應上，D258A分別比野生型小318及108倍。由D258A的pH profile為一鐘型圖及沒有明顯同位素效應的結果顯示D258在蘋果酸酶的催化反應中並不是唯一的催化鹼，可能另有一殘基在D258A酶分子的反應中扮演催化鹼的角色。而D258可能只是和金屬離子配位結合且極化金屬，而極化的金屬將加速酶分子氧化還原反應中氫質子的轉移。

Pigeon liver malic enzyme catalyzed the reversible oxidative decarboxylation of L-malate to produce pyruvate and CO2 in the presence of divalent metal ion (Mg+2 or Mn+2), with concomitant reduction of NADP+ to NADPH. Malic enzyme was proposed to follow a general acid-base catalysis mechanism. From the crystal structure and metal catalyzed oxidation studies, D234, D235 and D258 were identified as the metal binding ligand in the pigeon liver malic enzyme. In the studies of Ascaris suum malic enzyme, D295 (corresponding to D258 in pigeon liver malic enzyme) was suggested to function as a general base in the catalysis. Moreover, from the crystal structure and mutation studies K162 were suggested that K162 was a general acid in the catalytic mechanism.
In order to further examine the roles of these two amino acid residues in enzyme catalysis, K162 and D258 were converted to alanine for detailed kinetic studies. Initial velocity results showed that the kcat values of K162A and D258A was decreased by 26300 and 7700 fold, respectively, to that of wild type. The activity of K162A mutant was insensitive to TNBS chemical modification for 20 min. For the pyruvate reduction reaction, the kcat value of K162A was identical to that of the wild type. The kcat value of K162A for the oxalacetate decarboxylative reaction was reduced by 3262 fold. The rH value of K162A was reduced 6.5 fold to that of the wild type. In the pH profile studies, K162A showed pH-independence pattern for kcat value at high pH. Therefore we suggest that the ε-amino group of K162 may act as a general acid, donating a proton to the 3-carbon of enolpyruvate to give ketopyruvate.
For D258A mutant, the kcat value for two partial reactions were reduced by 318 and 108 fold to those of the wild type, respectively. The pH profile of D258A is identical to wild type showed a bell-shaped curve. There is no obviously deuterium isotope effect for D258A . Therefore we suggest that a second amino acid residue might act as a general base in D258A mutant to complete the enzymetic reaction. However, the D285 residue may not participate in general acid-base mechanism. It may only act as a chelator to polarize metal ion to facilitate the transfer of hydride ion in the oxidoreductive reaction.

目 錄
頁次
縮寫表………………………………………………………………. II
表目錄……………………………………………………………….III
圖目錄……………………………………………………………….IV
中文摘要……………………………………………………………..V
英文摘要…………………………………………………………….VI
壹、緒論…………………………………………………………….. 1
貳、實驗材料與方法……………………………………………….. 6
參、實驗結果……………………………………………………….22
肆、討論…………………………………………………………….28
表…………………………………………………………………….38
圖…………………………………………………………………….44
參考文獻…………………………………………………………….54

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34. 陳威男：鴿肝蘋果酸酶234殘基在金屬結合位置的研究，2001
35. 張碩欽：鴿肝蘋果酸酶催化上天們冬胺酸235、257、258功能之研究，2001