血栓素合成酶之純化、結晶及結構分析

Abstract

血栓素合成酶 (thromboxane A2 synthase, TXAS) 與前列環素合成酶 (prostacyclin synthase, PGIS) 屬於帶有血基質 (heme) 的細胞色素P450 (cytochrome P450) 蛋白家族的成員。前者可將前列腺素H2 (prostaglandin H2, PGH2) 異構化，以生成血栓素A2 (thromboxane A2, TXA2)。前列環素合成酶則可使前列腺素H2異構化為前列環素 (prostacyclin, PGI2)。雖然此二酵素皆以前列腺素H2為受質，但產物的生理活性卻互為拮抗，血栓素A2會造成血管收縮與血小板凝集，前列環素則會使血管舒張並抑制凝血。因此這兩種分子在生物體內的比例與心血管系統的運作密切相關；過多的血栓素A2會造成心臟病、中風及血栓等心血管疾病。相反地，若是前列環素過多則會導致一些發炎反應。由之前的研究發現：血栓素合成酶與受質鍵結的位置是在C9-O上，而前列環素合成酶則是鍵結在受質的C11-O上，此差異使得氧自由基形成的位置不同，因而生成不同的產物。 為了探討血栓素合成酶與前列環素合成酶與受質結合之立體特異性，本實驗室已得到前列環素合成酶的晶體結構。而本研究的主要目的就是得到大量且純度高的血栓素合成酶進行結晶學研究並嘗試利用同源蛋白結構模擬找出可能影響受質立體選擇性的胺基酸，再和前列環素合成酶比較以探討活性中心附近之結構差異。此外，若能取得血栓素合成酶的結構，勢必能有效地設計出針對血栓素合成酶專一性抑制的心血管疾病治療藥物。 目前為止人類血栓素合成酶利用基因重組去除N端穿膜序列及使用介面活性劑萃取後，已經能夠取得足量且純度高的蛋白，但是在緩衝液中的穩定性仍欠佳，以致於尚未得到良好的晶體以進行X光繞射分析。雖然如此，利用同源蛋白結構模擬，我們仍然預測了在血栓素合成酶與前列環素合成酶中可能對於受質造成立體選擇性的原因，而這些預測結果或許能利用點突變及活性分析加以證實。

Thromboxane A2 synthase (TXAS) and prostacyclin synthase (PGIS) belong to the heme-containing cytochrome P450 (CYP) enzyme superfamily. TXAS catalyzes an isomerization of prostaglandin H2 (PGH2, an endoperoxide) to form thromboxane A2 (TXA2). On the other hand, PGIS converts the same substrate into a differ product named prostacyclin (PGI2). TXA2 promotes vasoconstriction and platelet aggregation through binding to the TXA2 receptor. The biological functions of TXA2 are antagonized by PGI2, which serves as a potent vasodilator and anticoagulator. The proper balance between TXA2 and PGI2 is crucial for regulating the homeostasis of cardiovascular system. Excess amount of TXA2 is linked to heart attack, stroke, and thrombosis, whereas increasing PGI2 would cause inflammatory effects. Interestingly, both TXA2 and PGI2 are isomers of PGH2, and the isomerization reactions of PGH2 to either PGI2 or TXA2 start from a cleavage of the endoperoxide moiety (the C9-O-O-C11 group) of PGH2. Earlier spectroscopic analysis using a series of PGH2 analogs has revealed that, although TXAS and PGIS both cleave the endoperoxide, they bind PGH2 with stereo-specific distinction: the C9-O and C11-O of PGH2 serve as the heme ligand for TXAS and PGIS, respectively. Our lab has previously determined the crystal structure of PGIS, in this study we hope to understand the structural basis of binding selectivity of TXAS by X-ray crystallography or homology modeling combining with site-directed mutagenesis and kinetic analysis. Furthermore, the crystal structure of TXAS should facilitate the development of small molecule inhibitors which maybe useful for treating cardiovascular diseases. The recombinant human TXAS protein can be expressed and extracted by detergents, and we are attempting to obtain large amount of high purity protein sample for crystallization. In addition, we have used homology modeling to construct structural model of TXAS and compared it with crystal structure of PGIS by superimposition. This analysis has revealed potential structural differences in the substrate binding pocket that might be important in defining substrate binding selectivity. Site-directed mutagenesis and kinetic analysis will be performed to confirm the functional significance of our observations.