- 學位論文
人類α-甲基醯基輔酶A消旋酶之催化機制及疾病相關突變的結構闡述
Structural elucidation of human α-methylacyl-CoA racemase provides insight into its catalytic mechanism and disease-associated mutations
摘要
α-甲基醯基輔酶A 消旋酶（α-methylacyl-CoA racemase, AMACR）被發現在前列腺癌細胞中大量表現，並已作為診斷的生物標記。此酵素參與人體中支鏈脂肪酸的代謝途徑，負責支鏈脂肪酸中Cα的手性反轉，其受質包含攝食紅肉中富含的支鏈脂肪酸、藥物（如布洛芬, ibuprofen）或人體內源性的膽酸前驅物。目前已知降低AMACR 表現量有助於減緩癌細胞增生速率，因此被認為是有潛力的藥物標靶。此外，AMACR 的單一核苷酸變異(SNP)造成S52P 和L107P 的突變也使得酵素功能缺失，而導致神經性病變的症狀。由於缺乏蛋白質結構的資訊，SNP 與蛋白功能的相關性也尚未明瞭。 透過蛋白質晶體學實驗，我們已順利地解出人類AMACR 結構，及其與受質類似物Isobutytryl CoA (IBCoA)與酵素共結晶的複合體結構，藉此暸解受質結合區域及酵素催化機制。結構分析可發現，H122 和D152 為活性殘基，而受質的thioester oxygen 可與酵素D123 殘基骨幹的氮形成氫鍵。而AMACR 的SNP 研究發現的兩個致病突變S52P、︑L107P，實驗分析發現可能會影響蛋白質穩定性，同時S52P、 L107P 位置接近受質結合位置，可能也會影響受質的結合。我們也進行兩個已發表之非競爭型AMACR 抑制劑Ebselen、Ebselen oxide 的研究，實驗顯示Ebselen和Ebselen oxide 都會降低蛋白質的穩定性。從晶體結構觀察Ebselen、Ebselen oxie都會使C117 附近殘基的結構失序(disorder)，推測其作用機制為選擇性共價性修飾，進而造成蛋白質穩定性下降並失去活性。本篇研究提供人類AMACR 結構，探討其反應機制及疾病相關突變S52P 和L107P 的結構闡述，並推測非競爭型抑制劑的作用機制，希望能提供未來藥物設計的結構依據。
並列摘要
AMACR (α-methylacyl-CoA racemase), catalyzing a key chiral inversion step in metabolism of branched-chain fatty acid, ibuprofen and related drugs, was overproduced in prostate cancer and has been used as a prostate cancer biomarker. Previous studies shows that decreasing the expression of AMACR through using short interfering RNA constructs can slow the growth of prostate cancer cell lines, indicating that AMACR may be a new treatment for cancers. In addition, AMACR is associated with human diseases because of function deficiency caused by single base mutation S52P and L107P. Due to the lack of any experimentally determined structure of the human AMACR, the roles of SNP correlated to protein functions remains unclear. Here, the crystal structure of human AMACR and the complex structure with Isobutytryl CoA (IBCoA), an structure analogue, were determined, showing that the catalytic residues are H122 and D152, also showing the thioester oxygen hydrogen bonded to the backbone amide proton of D123. We fine that pathogenic mutants L107P and S52P are unstable than the wild type, and both mutants located very closed to the binding pocket. Ebselen and ebslene oxide, two reported non-competitive AMACR inhibitors, decrease the Tm and disordered the residues structure near C117, probably specific covalent bond to C117 then make structure change. The structures of human AMACR provide an attractive structural basis of substrate recognition for further drug design.