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標題: 決定人類抗酶抑制子二聚體結構的關鍵因子
Critical Factors Determining the Dimeric Structure Formation of Human Antizyme Inhibitor
作者: 蘇國良
Su, Kuo-Liang
關鍵字: ornithine decarboxylase
antizyme inhibitor
analytical ultracentrifugation
quaternary structure
出版社: 生命科學系所
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摘要: Chinese Abstract中文摘要 人類鳥胺酸脫羧酶(ornithine decarboxylase,ODC,EC為依賴5’-磷酸吡多醛 (pyrodoxal 5’-phosphate,PLP)輔酶之酵素,主要催化體內多元胺合成起始與速率決定步驟的酵素。此酵素以二聚體型式存在,需兩個鳥胺酸脫羧酶的單體活性中心聚合形成同源二聚體。抗酶(antizyme,AZ)可以跟鳥胺酸脫羧酶結合,形成異源二聚體,使鳥胺酸脫羧酶被26S蛋白解體降解,並不需要先跟泛素結合。人類抗酶抑制子(human antizyme inhibitor,AZI)以單體存在,與鳥胺酸脫羧酶屬高度同源的蛋白但不具有酵素活性,抗酶抑制子與抗酶的結合能力大於鳥胺酸脫羧酶,因此可以回復鳥胺酸脫羧酶的活性。我們的研究目標是經由探討鳥胺酸脫羧酶與抗酶抑制子的四級結構來確認重要的胺基酸位置。透過鳥胺酸脫羧酶與抗酶抑制子的序列比對,找出形成二聚體胺基酸位置的不同,將抗酶抑制子序列(S277、S331、E332及D389)改成鳥胺酸脫羧酶的序列(R277、Y331、D332及Y389),這些突變種利用分析級超高速離心機來分析沉降係數大小分布。從沉降係數及連續性大小分布分析可以得到野生種鳥胺酸脫羧酶是以二聚體分布。而從自我單體二聚體結合分析得到解離常數為0.18 μM。相對的野生種抗酶抑制子呈現單體存在而解離常數為84.0 μM。AZI-S331Y突變種呈現在單體及二聚體平衡中,解離常數為40.7 μM明顯比野生種低。AZI-S331Y-D389Y、AZI-S331Y-D389Y-S277R及AZI-S331Y-D389Y-S277R-E332D均為二聚體分布其解離常數分別為2.7 μM、1.3 μM及0.10 μM,很接近於野生種鳥胺酸脫羧酶。然而各種抗酶抑制子突變種如果沒有S331Y這個點突變,解離常數跟野生種抗酶抑制子並沒有差異。我們的研究成果發現胺基酸331這個位置是鳥胺酸脫羧酶及抗酶抑制子形成二聚體最主要的關鍵因子。
English Abstract Human ornithine decarboxylase (ODC; EC is a pyrodoxal 5'-phosphate (PLP) dependent enzyme that catalyzes the first and rate-limiting step in polyamine biosynthesis. The enzyme is an obligate homodimer, and the two identical active sites are formed at the dimer interface between the monomers. Antizyme (AZ) interacts with ODC to form heterodimer that is then degraded by 26S proteasome without ubiquitin binding. Human antizyme inhibitor (AZI) is highly homologous to ODC but lacks enzymatic activity, and it exists as a monomer. AZI has higher affinity toward AZ than ODC and that may rescue ODC enzyme activity. In this study, we aim to identify the essential amino acid residues governing the quaternary structure formation for ODC and AZI. Based on the multiple sequence alignments of ODC and AZI, the non-conserved amino acid residues in the putative dimer-interface of AZI (S277, S331, E332 and D389) were changed to the amino acid residues of ODC (R277, Y331, D332 and Y389, respectively). Analytical ultracentrifugation were used to obtain the size-distribution of these AZI mutants. Sedimentation velocity and continuous size distribution analysis demonstrated that the wild-type ODC (ODC-wt) is a dimer with a disassociation constant (Kd) of 0.18 μM. In contrast, the wild-type AZI (AZI-wt) displayed as a monomer with a Kd of 84.0 μM. The AZI-S331Y mutant displayed a monomer-dimer equilibrium with a Kd of 40.7 μM showing a lower Kd than that of AZI-wt. The double mutant, AZI-S331Y-D389Y, triple mutant, AZI-S331Y-D389Y-S277R and quadruple mutant, AZI-S331Y-D389Y-S277R-E332D displayed as a dimer with a Kd value of 2.7 μM, 1.3 μM and 0.10 μM, respectively, close to that of ODC-wt. The AZI-mutants without S331Y showed similar Kd values to that of AZI-wt. Our findings indicated that the residue 331 play a key role determining the dimer formation of ODC and AZI.
其他識別: U0005-2207200915313400
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