Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23078
標題: 人類蘋果酸酶及抗酶結構與功能關係之探討
Studies on Structure and Function Relationship of Human Malic Enzyme and Antizyme
作者: 謝如怡
Hsieh, Ju-Yi
關鍵字: malic enzyme
蘋果酸酶鳥胺酸脫羧酶抗酶抑制因子
ornithine decarboxylase
antizyme inhibitor
antizyme
polyamine
抗酶多元胺
出版社: 生命科學系所
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摘要: 1. 蘋果酸酶催化氧化脫羧反應使蘋果酸轉變為丙酮酸,並伴隨著NAD(P)+轉變為NAD(P)H的還原反應。 哺乳動物細胞中具有三種異構型,粒線體 (m) 及細胞質 (c) 中只利用NADP+為輔酶的蘋果酸酶 (m-NADP-ME 和c-NADP-ME)),及粒線體中可同時利用NAD+和NADP+為輔酶的蘋果酸酶 (m-NAD(P)-ME),但生理換境下偏好NAD+。 在這個研究中, 我們說明了在人類粒線體蘋果酸酶 (m-NAD(P)-M) 中調控輔酶專一性的決定性胺基酸 (346、347和362) 成功地轉移了輔酶偏好而成為一個以NADP+為主的酵素。 在輔酶偏好轉移中,最重要的胺基酸是362,且所有含有Q362K的突變株均具有較大的反應速率 (kcat,NADP,以NADP+做為輔酶的反應速率)。 K346S和Y347K突變則扮演著輔助的角色在於增強輔酶轉移的能力,而Y347K的影響力遠重要於K346S。 另外,K346S 明顯地降低ATP的抑制能力並具有一個較大的Ki,ATP值,這說明了 346在 ATP異構型專一抑制性的重要。 同時突變株喪失了蘋果酸的協同作用及反丁烯二酸的異位調控。 整體而言, 我們以酵素動力學的證據來驗證改變輔酶偏好性的調控因素,並找到K346S/Y347K/Q362K m-NAD(P)-ME為最有效力的突變株。 2. 多元胺 (polyamines) 是可通透細胞的正電小分子可調控核酸遺傳物質DNA、RNA及蛋白質,並牽涉到細胞週期、細胞調亡、蛋白質合成以及細胞增生和癌化轉形。鳥胺酸脫羧酶 (ornithine decarboxylase,ODC) 是一個依賴5’-磷酸吡多醛 (pyrodoxal 5’-phosphate,PLP) 輔酶之酵素,催化鳥胺酸 (ornithine) 轉變成腐胺 (putrescine) 並且是一個調控體內多元胺平衡之速率限制步驟。人類鳥胺酸脫羧酶是一個短生命週期的蛋白質,受到抗酶 (antizyme,AZ) 調控降解 (degradation) 而不需要泛素 (ubiquitin) 參與。 抗酶可以和鳥胺酸脫羧酶形成異源雙聚體 (heterodimer) 並降低鳥胺酸脫羧酶同源雙聚體 (homodimer) 形成而抑制了鳥胺酸脫羧酶的酵素活性。 抗酶抑制因子 (antizyme inhibitor) 是鳥胺酸脫羧酶的同源蛋白質且不具有任何酵素活性,並且相較於鳥胺酸脫羧酶有較高的親和性去抑制鳥胺酸脫羧酶和抗酶的異源雙聚體形成,所以鳥胺酸脫羧酶可再次回復酵素活性並能再次提供腐胺並有效調控多元胺。 在這研究中,我們把目標放在鳥胺酸脫羧酶和抗酶間的交互作用,並且探究人類抗酶結合位上之結合區域及重要胺基酸,並確認在抗酶抑制因子與抗酶結合區是否具有相同的交互作用。我們也可以說明抗酶結合位是否能調控鳥胺酸脫羧酶的四級結構變化,和藉由找到此段抗酶結合位來設計有效性胜肽型抗癌藥物作為鳥胺酸脫羧酶及多元胺相關之癌症治療。
1.Malic enzyme (ME) is a oxidative decarboxylase that catalysis a reversible reaction of malate to pyruvate, accompanying a reduction of NAD(P)+ to NADPH. There are three isoforms in mammalian cells, both mitochondria and cytosolic NADP+-dependent MEs (m-NADP-ME and c-NADP-ME) utilize NADP+ as the cofactor, and m-NAD(P)-ME is unique isoform that displayed a dual cofactor specificity, but prefers NAD+ under physiological condition. In this studies, we demonstrated the decisive amino acids (346, 347 and 362) in human m-NAD(P)-ME to govern the cofactor specificity, and successfully shifted the cofactor preference to be a NADP+-dependent enzyme. Most considerable residue for cofactor shifting was 362, and all mutants containing Q362K possessed a greater kcat,NADP value in comparison of the kcat,NAD value. The mutation of K346S and Y347K played the assistant role to promote the capacity of cofactor-shifting, and the influence of Y347K was more important than that of K346S. Additionally, the mutant K346S obviously decreased the inhibition of ATP, with a large Ki,ATP value, suggesting that residue 346 in human m-NAD(P)-ME is significant for the isoform-specific ATP inhibition. Non-cooperativity of malate binding and non-allosteric fumarate activation were other characters for m-NAD(P)-ME mutants. Overall, we provided the kinetic evidences to examine the regulative factors for cofactor specificity, and K346S/Y347K/Q362K m-NAD(P)-ME was the powerful mutant in our studies. 2.Polyamines (putrescine, spermidine, and spermine) are small, positively charged molecules that permit cells to regulate DNA, RNA, and protein involved in the cell cycle, apoptosis, protein synthesis, cell proliferation and neoplastic transformation. Ornithine decarboxylase (ODC), a pyridoxal phosphate (PLP)-dependent enzyme, catalyzes the transformation of ornithine to putrescine and is a rate-limiting step for polyamine homeostasis. Human ODC is a short-lived protein controlled by antizyme (AZ) with ubiquitin-independent degradation. AZ can interact with ODC to form an AZ/ODC heterodimer, diminishing ODC homodimer formation and inhibiting ODC enzyme activity. Antizyme inhibitor (AZI) is a homologous protein of ODC without any decarboxylating activity, and it has higher affinity than ODC to interact with AZ and to restrict AZ/ODC heterodimer formation. Therefore, ODC can recover its enzyme activity and supply putrescine again, effectively regulating polyamine. In this study, we have focused on the interaction between ODC and AZ with different lengths of AZ segments to delineate the important regions and dominant amino acids in human AZ binding site, and to confirm that AZI is behaving in the same fashion with the major binding region of AZ. We can also elucidate whether the AZ binding region commands the ODC quaternary structure or not. Finally, we will use the AZ binding region to design a functional peptide drug for antineuoplatic therapy in connection with ODC and polyamines.
URI: http://hdl.handle.net/11455/23078
其他識別: U0005-2007201011163800
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2007201011163800
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