Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22891
標題: 探討人類第四型胜肽精胺酸去亞胺酶的四級結構組成與酵素催化之間關係
Study on the relationship between the quaternary structure organizations and enzyme catalysis of human peptidylarginine deiminase 4 (PAD4)
作者: 江玉秀
Chiang, Yu-Hsiu
關鍵字: peptidylarginine deiminase 4
第四型胜肽精胺酸去亞胺酶類風濕性關節炎
PAD4
rheumatoid arthritis
quaternary structure organizations
enzyme catalysis
四級結構
酵素催化活性
出版社: 生命科學系所
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摘要: 胜肽精胺酸去亞胺酶 (peptidylarginine deiminase, PAD) 是一種需要鈣離子的酵素,負責執行將鍵結在蛋白質上面的精胺酸 (arginine) 轉變為瓜胺酸 (citrulline) 的protein citrullination (deimination) 的過程。PAD4是這裡面唯一的一種帶有nuclear localization signal (NLS) 區域的異構型,且PAD4的基因是一個對於類風濕性關節炎易感受性的基因座。PAD4在沒有結合鈣離子或是有結合鈣離子和受質狀態下的結構已經藉由X-光晶體繞射學被解出來。PAD4的結構呈現由兩個單體分子以頭跟尾互相接觸的方式形成雙體分子存在。然而,對於PAD4的酵素調控和單體與單體之間的交互作用關係仍然不清楚。為了獲得PAD4四級結構穩定性的知識,我們使用LIGPLOT和DIMPLOT找出在PAD4的兩個單體結合介面上擁有氫鍵交互作用的重要胺基酸。其中,在一個單體上面的Arg8與另一個單體上的Asp547有氫鍵作用,且此兩個胺基酸皆為帶有電性胺基酸,存在有離子鍵交互作用。另一方面,在一個單體上面的Tyr435亦與另一個單體上的Tyr237與Glu281有氫鍵交互作用。之後,我們利用定點突變方法創造一系列位在兩個單體結合介面上的Arg8,Tyr237,Glu281,Tyr435和Asp547等位置的胺基酸突變去破壞雙體結構,結合分析型超高速離心機 (analytical ultracentrifugation) 之技術來分析四級結構狀態,並且進行酵素活性測定。結果顯示,有造成四級結構改變的PAD4突變,其活性以及協同作用比起野生型 (wild-type) 的PAD4有伴隨著降低的現象。這些結果指出PAD4的雙體狀態不只對於維持整體結構的穩定性是重要的,同時也對酵素催化的調節方面是有影響力的。此外,Arg8與Asp547之間的離子鍵作用力只有輕微的影響兩個單體結合介面,而Tyr435上面的疏水性作用力則是個對於PAD4結構穩定性和活性上很重要的因子。
The peptidylarginine deiminase (PAD) is a Ca2+-dependent enzyme that can carry out protein citrullination (deimination) which is the conversion of protein-bound arginine to citrulline (Arg -> Cit). PAD4 is the only one isotype with a nuclear localization signal (NLS) region and the gene of PAD4 has a susceptible locus for rheumatoid arthritis (RA). The structures of PAD4 combined without or with calcium ions and the substrate have been resolved by X-ray crystallography. PAD4 is a dimer consisted of two monomer head-to-tail contact. However, it is still unclear the relationship between enzyme regulation and subunit-subunit interaction of PAD4. To gain insight into the quaternary structure stability of PAD4, we used LIGPLOT and DIMPLOT to find the important residues at dimer interface of PAD4 with hydrogen-bonded interaction. The Arg8 of one monomer is hydrogen-bonded with Asp547 of the other monomer and these two amino acid residues are charged residues containing the ionic interaction. On the other hand, the Tyr435 of one PAD4 monomer also has the hydrogen-bonded interaction with Tyr237 and Glu281 on the other monomer. Then we analyzed a series of PAD4 mutations at residues Arg8, Tyr237, Glu281, Tyr435, and Asp547 on the dimer interface by site-directed mutagenesis to disrupt the dimer organization combined with the analytical ultracentrifugation data and kinetic analysis. The mutations of PAD4 dimer interface caused quaternary structural change accompanied by the decreased activities and Hill coefficient (h) compared to WT. Here, our data indicated that the role of the dimeric state of PAD4 is not only important to maintain the stability of the overall structure but also to regulate its catalysis. Moreover, the ionic interaction between Arg8 and Asp547 slightly affect the dimer interface and the hydrophobic interaction of Tyr435 is the most important factor for the structure stability and activity of PAD4.
URI: http://hdl.handle.net/11455/22891
其他識別: U0005-1507200915120600
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1507200915120600
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