Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22278
標題: 第四型胜肽精胺酸去亞胺酶之活性探討
Characterization of the Enzymatic Activity of Peptidylarginine Deiminase 4
作者: 謝慧潔
Hsieh, Hui-Chieh
關鍵字: Peptidylarginine Deiminase 4;第四型胜肽精胺酸去亞胺酶
出版社: 生命科學系所
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摘要: 
第四型胜肽精胺酸去亞胺酶(Peptidylarginine deiminase 4, 簡稱PAD4)是一種和類風濕性關節炎之間具有高度相關性的酵素,其必須於二價鈣離子存在之生理條件下,始具有活性。而在前人發現的PAD4基因序列上之單一核苷酸多型性(Single nucleotide polymorphisms, 簡稱SNPs)中,有四個位點會被轉譯成蛋白質﹔其中的三個位點更牽涉到胺基酸改變,分別為﹕S55G, A82V, A112G。我們做了許多實驗用以探討SNPs對PAD4的影響。而從動力學實驗結果可以發現,A112G酵素之kcat值是重組野生型的兩倍,顯示此處可能為操控PAD4催化能力之重要位置。除此之外,S55G/A112G 和A82V/A112G 以及S55G/A82V/A112G酵素的kcat值也皆明顯大於重組野生型的kcat值,證明A112G位點突變確實會導致PAD4酵素活性提升。而在Ca2+活化實驗方面,不同於重組野生型所繪出的雙曲線圖,S55G, A112G, S55G/A112G和A82V/A112G以及S55G/A82V/A112G酵素之活化程度呈現出雙位相的表現。結果顯示,上述五種SNPs酵素,其蛋白結構似乎對Ca2+有兩種不同的親合力。我們另外使用與Ca2+化學特性相似的Mg2+做酵素活性抑制實驗,結果發現,這五種蛋白質中帶有A112G突變點的酵素(A112G, S55G/A112G, A82V/A112G, S55G/A82V/A112G),其活性相對於重組野生型較不易受Mg2+影響
。此外,為了更進一步探求SNPs酵素和重組野生型在二價金屬結合位置上的特性,我們還利用螢光光譜儀測定PAD4的三級結構。整合上述各種實驗所得到的結果,可以推知帶有A112G突變點的酵素,可能藉由Ca2+對其不正常的活化,促
使類風濕性關節炎的發生。

Peptidylarginine deiminase 4 (PAD4) is a Ca2+-dependent enzyme. The enzyme is highly correlated with rheumatoid arthritis. PAD4 gene has the single nucleotide polymorphisms (SNPs), four are exonic and three of them involved in amino acid substitutions: S55G, A82V, and A112G. We have established a series of SNP enzymes to identify the effects of SNPs on the PAD enzymes. The kcat value of A112G enzyme was higher than that of the wild-type enzyme by 2-fold, indicating that replacement of this residue elevated the catalytic capability of the enzyme. Notably, the kcat values of S55G/A112G and A82V/A112G and S55G/A82V/A112G were larger than that of wild-type enzyme, indicating that the A112G replacement causes the PAD enzyme more active. The activities of PAD4 were significantly stimulated by Ca2+ in a hyperbolic manner. Interestingly, the IC50 titration curves of Ca2+ for S55G, A112G, S55G/A112G, A82V/A112G, as well as S55G/A82V/A112G, displayed a biphasic curve indicating these SNP enzymes had two different binding affinities toward Ca2+. We also evaluated the Mg2+ inhibition to the PAD4 activity, and the results indicated the activities of these A112G-substituting enzymes were slighter affected by Mg2+ than the others. Furthermore, the enzyme conformations were analyzed with fluorescence quenching studies. Collectively, these findings suggest that the abnormal stimulation by Ca2+ for the enzyme activities of these A112G-substituting enzymes may be related to the
pathogenesis of rheumatoid arthritis.
URI: http://hdl.handle.net/11455/22278
其他識別: U0005-0207200611414800
Appears in Collections:生命科學系所

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