Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21234
標題: 第三,四,五號鈣離子結合位在人類第四型胜肽精胺酸去亞胺酶中所扮演的角色
Functional Characterization of Calcium Binding Sites 3, 4, 5 in Peptidylarginine Deiminase type 4
作者: 張家維
Chang, Chia-Wei
關鍵字: Peptidylarginine Deiminase type 4;第四型胜肽;精胺酸去亞胺酶
出版社: 生命科學系所
摘要: 
胜肽精胺酸去亞胺酶 (Peptidylarginine deiminase)是一種後轉譯修飾的酵素,在有鈣離子的情況下可以將蛋白質上的精胺酸 (arginine)轉變為瓜胺酸 (citrulline),胜肽精胺酸去亞胺酶總共有五種異構型 (isoforms),其中第四型胜肽精胺酸去亞胺酶近年來被認為是偵測類風濕性關節炎的重要因子。到目前為止,只有第四型胜肽精胺酸去亞胺酶的結構被結晶出來,從蛋白結構中可以知道第四型胜肽精胺酸去亞胺酶有五個鈣離子的結合位,分別為將這些鈣離子簡稱為Ca1、Ca2、Ca3、Ca4、Ca5,酵素在鈣離子的結合之下會促使結構的變化,使催化中心區域可以跟受質結合。過去的研究已知,Ca1和Ca2的結合位置鄰近催化中心並且會協助催化中心的形成,然而Ca3、Ca4、Ca5的功能目前還不確定,所以我們利用胺基酸點突變技術 (site-directed mutagenesis)配合酵素動力學實驗,針對Ca3、Ca4、Ca5結合位附近的胺基酸做研究,這些胺基酸分別為N153、D155、D157、D165、D168、E170、D176、D179、E252、D388,將這些胺基酸分別置換為丙胺酸 (alanine)或是結構相近但不帶電的醯胺胺基酸 (amide amino acid)。從結果可以發現,Ca5結合位的重要性沒有Ca3、Ca4結合位來的明顯,即使將Ca5結合位完全去除,酵素仍保有一半左右的活性,然而Ca3和Ca4結合位的破壞,會導致酵素活性大為減低;從蛋白質螢光 (fluorescence)的實驗中,我們也得知Ca3、Ca4、Ca5結合位的胺基酸對於酵素的三級結構有不同程度的影響性,這也間接解釋了Ca3、Ca4、Ca5結合位對於酵素結構的重要性。總結以上的實驗,我們可以知道雖然Ca3、Ca4、Ca5結合位不像Ca1、Ca2結合位這麼接近活性中心,但是Ca3、Ca4、Ca5結合位的存在,對於酵素的三級結構及活性都有一定程度上的影響。

Peptidylarginine deiminase (PAD) is a post-translational modifying enzyme which catalyzes the conversion of protein arginine to citrulline in the presence of calcium. The PAD family has five isoforms and one of them, PAD4, is considered as the important factor of rheumatoid arthritis. The structures of PAD4 without or with calcium ions have been resolved by X-ray crystallography. There are five calcium ions occupying on the enzyme that are designated Ca1, Ca2, Ca3, Ca4, and Ca5, respectively. Calcium-binding induces PAD4 conformational change to form the active-site cleft. The Ca1 and Ca2, which are near to the catalytic site, are defined as critical calcium ions that assist in recognition of the substrate. The other three calcium ions are in the N-terminal domain away from the catalytic site. The functional roles of Ca3, Ca4, and Ca5 are still undefined. Here we use site-directed mutagenesis and enzyme kinetic analysis to interpret the role of Ca3, Ca4, and Ca5 binding sites. The ten residues, N153, D155, D157, D165, D168, E170, D176, D179, E252, and D388, are replaced by alanine or amide amino acid to interrupt the calcium binding on this region. These mutants at the Ca3 and Ca4 binding sites show higher Km and lower kcat than WT, suggesting that Ca3 and Ca4 are essential for PAD4 catalysis. Our fluorescence studies further demonstrate that these mutants display different conformations as compared with WT. The mutants of D168, E170, and E252, which participate the Ca5 binding, have weaker effects on Km and lower kcat than Ca3 and Ca4. Our findings show that Ca3 and Ca4, although far away from the active site, still have significant influence on substrate binding and calcium cooperativity.
URI: http://hdl.handle.net/11455/21234
Appears in Collections:生命科學系所

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