血栓素合成酶與前列環素合成酶之結構分析

Structural Analysis of Thromboxane A2 Synthase and Prostacyclin Synthase

葉君毅(Chun-Yi Yeh)

指導教授 ： 詹迺立

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201600549

摘要

世界衛生組織之統計顯示，世界前十大死因中，心血管相關疾病就佔了三項，因此若能有效的治療與控制心血管疾病，勢必是人類的一大福音。心血管疾病與體內前列腺素H2 (prostaglandin H2, PGH2) 及其下游產物 (prostanoids) 的含量與生物活性是息息相關的，其中血栓素合成酶(thromboxane A2 synthase, TXAS) 和前列環素合成酶(prostacyclin synthase, PGIS) 是調控心血管功能的要角。血栓素合成酶可以把前列腺素H2催化為血栓素(thromboxane A2, TXA2)，而前列環素合成酶則可以把前列腺素H2催化為前列環素(prostacyclin, PGI2)，催化機制是利用血基質上的鐵離子辨認並與前列腺素H2之內過氧化基團(endoperoxide) 中不同位置的氧結合(血栓素合成酶辨識C9-O；前列環素合成酶辨識C11-O)，再經由血基質引發的O–O鍵裂解，合成不同的產物。血栓素是個非常有效的血管收縮劑，並可誘發血小板的聚集與釋放，而形成血栓；前列環素則恰好與之拮抗，可使血管舒張及抑制血小板凝集。本研究希望可以藉由解析此兩種酵素的晶體結構，而了解二者催化專一性的結構基礎。
我們先使用 E. coli 大量表達血栓素合成酶和前列環素合成酶，並使用液相層析法進行蛋白純化，接著利用受質前列腺素H2 的類似物U51605 與兩種酵素結合，進行複合體的結晶嘗試，目前已順利解出人類前列環素合成酶與U51605 形成的複合體結構，發現U51605 結合可改變血基質之丙酸基(propionate groups) 與酵素活性中心附近鹼性胺基酸的交互作用，先前本實驗室亦曾在斑馬魚前列環素合成酶的結構中觀察到此種構形變化，推測應與酵素催化電子傳遞反應與O–O 鍵裂解的效率有關。目前也順利發現血栓素合成酶的結晶條件，並積極進行血栓素合成酶與U51605、U44069 等受質類似物形成之複合體的結構解析，期待藉由比較二者活性中心與受質結合的異同，輔助血栓素合成酶專一性抑制劑的開發。

關鍵字

血栓素合成酶(CYP5A1) ； 前列環素合成酶(CYP8A1) ； 細胞色素P450 ； 心血管疾病 ； 蛋白質結晶 ； X-ray 繞射分析

並列摘要

Statistics conducted by the World Health Organization reveals that cardiovascular-related diseases claim three spots among the top 10 causes of death. Therefore, it is crucial to develop new diagnostic tools and effective treatments for these diseases. Previous studies provide a strong indication that the production and biological activities of prostaglandin H2 (PGH2) and its downstream prostanoids are linked closely to the cardiovascular functions. Using PGH2 as the common substrate, thromboxane A2 synthase (TXAS) and prostacyclin synthase (PGIS) catalyze respectively the production of thromboxane A2 (TXA2), a potent inducer of vasoconstriction and platelet aggregation, and prostacyclin (PGI2), a vasodilator and inhibitor of thrombosis. How mechanistically does these two enzymes use the same substrate but generate different products with completely opposite biological activities? Spectroscopic studies suggested that TXAS interacts with the C-9 oxygen of PGH2 using its ferric heme, whereas PGIS bind to the C-11 oxygen by its heme iron. The goal of this work is to provide a structural understanding on the strict catalytic stereo-specificity of these two synthases.
To this end, I have successfully determined the crystal structure of PGIS in complex with the substrate analog U51605. This result demonstrates a stereo-specific substrate binding and suggest structural features of the enzyme that may facilitate isomerization to produce PGI2. In addition, by employing a new purification protocol, I have prepared large amount of structurally and functionally intact TXAS and obtained diffracting crystals of this important enzyme. It is hopeful that the crystal structure of TXAS will be determined by X-ray crystallography in the near future to provide new insights into its catalytic mechanism. Moreover, with the crystal structures of both TXAS and PGIS available, it is possible to design small molecule inhibitors that specifically inhibit TXAS but not PGIS, for treating cardiovascular diseases.

並列關鍵字

thromboxane A2 synthase (TXAS, CYP5A1) ； prostacyclin synthase (PGIS, CYP8A1) ； cytochrome P450 ； cardiovascular vessel disease ； protein crystallography ； X-ray diffraction

參考文獻

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