Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21683
標題: 人類鳥胺酸脫羧酶與抗酶抑制子抗酶結合位上 對抗酶結合能力之探討
Functional characterication of the antizyme binding site on human ornithine decarboxylase and human antizyme inhibitor
作者: 劉儼嶔
Liu, Yen-Chin
關鍵字: ornithine decarboxylase
鳥胺酸脫羧酶
antizyme
Antizyme inhibitor
抗酶
抗酶
抑制子
出版社: 生命科學系所
摘要: 人類鳥胺酸脫羧酶(ornithine decarboxylase, ODC; EC 4. 1. 1. 17)是體內多 元胺(polyamines)生合成路徑中第一步驟與速率決定的酵素。ODC 為5’-磷酸 吡哆醛(PLP)依賴型酵素,催化過程結構上需形成雙聚體才能進一步催化鳥胺 酸(L-ornithine)釋出腐胺(putrescine)及CO2。ODC 為泛素(ubiquitination) 作用不依賴型酵素,抗酶(antizyme, AZ)能夠與鳥胺酸脫羧酶結合使其無法形 成雙聚體而失去其酵素活性,在細胞內與抗酶結合的鳥胺酸脫羧酶更容易被26S 蛋白酶體(proteasome)分解,而不需先與泛素結合。抗酶抑制子( Antizyme inhibitor, AZI)是序列及分子量都與鳥胺酸脫羧酶十分相似的蛋白質,但不具有 酵素活性。抗酶抑制子與抗酶的結合能力比鳥胺酸脫羧酶還強,能回復鳥胺酸脫 羧酶受抗酶抑制的情形。經序列比對,鳥胺酸脫羧酶與抗酶抑制子在抗酶結合位 上可能影響其結合能力的胺基酸有125、126、133、135、140 位置之不同。在活 性抑制的結果中,以ODC-N125K 及ODC-M140K 受抑制的情形較明顯,代表側 鏈上正電荷是決定鳥胺酸脫羧酶及抗酶抑制子與抗酶結合能力不同的關鍵。進一 步探討鳥胺酸脫羧酶突變型、抗酶抑制子突變型、抗酶三者間的交互作用,實驗 結果顯示突變型ODC-N125K 及ODC-M140K 與抗酶結合的親和性比鳥胺酸脫羧 酶野生型佳,突變型AZI-K125N 及AZI-K140M 則無法與鳥胺酸脫羧酶野生型競 爭抗酶,說明側鏈中的正電荷是影響與抗酶結合親和性的因素。探討ODC 與AZI 對AZ 的解離常數(Kd),結果顯示突變型ODC-N125K/ODC-M140K 的抗酶結合 親和性與抗酶抑制子野生型有相同程度的效果,證明正電荷為抗酶結合親和性的 影響關鍵因素。
Human ornithine decarboxylase (ODC; EC 4. 1. 1. 17) is the first and rate-limiting enzyme in the polyamine biosynthesis pathway. It is a pyridoxal 5'-phosphat (PLP)-dependent homodimeric enzyme that provides the only route for converting ornithine to putrescine and carbon dioxide. Antizyme (AZ) binds to ODC, inhibits ODC activity and promote the ubiquitin-independent degradation of ODC by 26S proteasome. Antizyme inhibitor (AZI) is highly homologous to ODC but is not enzymatically active. AZI, which has higher binding affinity to AZ, can rescue ODC from antizyme inhibition. Based on the sequence alignments of ODC and AZI, 125, 126, 133, 135, 140 residues are different in AZ-binding element between them. Our data indicated that the higher inhibited activities of ODC-N125K and ODC-M140K, which referred to the higher binding affinity to AZ, suggesting that the positive charge of side chain are crucial for AZ-binding affinity. Comparison with the dissociation constant of AZI-wt and ODC-N125K/M140K toward AZ, it indicated that ODC-N125K/M140K has a decreased Kd value similar to that of AZI, further supporting that the positive charge plays a crucial role in AZ-binding affinity.
URI: http://hdl.handle.net/11455/21683
Appears in Collections:生命科學系所

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