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標題: 人類第四型胜&;#32957;精胺酸去亞胺&;#37238;上五個鈣離子結合位之功能探討
Functional Characterization of Five Calcium Binding Sites in Peptidylarginine Deiminase Type 4
作者: 黃郁妮
Huang, Yu-Ni
關鍵字: peptidylarginine deiminase type 4;calcuim binding sites;circular dichroism;fluorescence
出版社: 生命科學系所
人類胜&;#32957;精胺酸去亞胺&;#37238;(human peptidylarginine deiminase, PAD)是一類在protein citrullination (deimination)中將鍵結在蛋白質上的精胺酸(arginine)轉變成瓜胺酸(citrulline)的酵素,且在鈣離子存在下才具有活性。此類胜&;#32957;精胺酸去亞胺&;#37238;有五種異構型(isoforms),其中PAD4被認為與偵測類風濕性關節炎因子有很密切關係。從結構來看可以發現有五個鈣離子結合位,分別將這些鈣離子以Ca1、Ca2、Ca3、Ca4及Ca5稱之。已知此酵素在與鈣離子結合後會引起結構改變使得催化中心區域可以與受質結合,且Ca1和Ca2的結合位至鄰近催化中心並協助催化中心的形成,相反的,其他三個位置功能則還不是非常清楚。為了探討這五個鈣離子結合位的重要性並比較,將附近的胺基酸利用點突變技術(site-directed mutageneis)全部置換成丙胺酸(alanine)配合酵素動力學實驗。結果顯示:Ca1、Ca2、Ca3、Ca4皆會影響活性,Ca5則相對沒那麼重要;從圓二色光譜儀(Circular Dichroism, CD )及蛋白質螢光(fluorescence)實驗中也得知Ca1、Ca2、Ca3、Ca4胺基酸對於酵素的二、三級結構都有不同程度的影響,甚至Ca3、Ca4對於結構影響程度大於Ca1、Ca2;熱穩定度實驗則解釋鈣離子對酵素結構的穩定性。總結以上實驗可以得知Ca3、Ca4結合位置不像Ca1、Ca2結合位置那麼靠近活性中心,但是重要性仍不容小覷。

Human peptidylarginine deiminase, PAD, is a kind of enzyme which can carry out protein citrullination (deimination) that, the conversion of peotein-bound arginine to citrulline (arginine → citrulline), working in presence of calcium. There are five isoform of PAD, one of them, PAD4, which is currently considered as the detection of rheumatiod arthritis factor. There are five calcium binding sites on PAD4 have been identified by X-ray crystallography, designated Ca1, Ca2, Ca3, Ca4 and Ca5, represently. Known that enzymes binding with calcium then induce conformational change to make substrate binding to catalytic site, Ca1 and Ca2 help it happened, but others function is not clearly. To understand the important and compare with these five calcium binding sites, we replaced all residues to alanine by site-directed mutageneis and analysis by enzyme kinetic assay. Results indicated Ca1, Ca2, Ca3 and Ca4 affect enzyme activity, Ca5 is lesser important. Circular dichroism and fluorescence data further demonstrate that these mutants display different conformations in second and third structure, even Ca3 and Ca4 are more influence than Ca1 and Ca2. In thermal denaturation experiment shown that calcium ions contributed to structural stability of enzyme. Beyond the discussed above, although Ca3 and Ca4 are not like Ca1 and Ca2 that near by active sites, still have significant influence.
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