血栓素合成酶與突變的前列環素合成酶之結構研究

Abstract

血栓素合成酶 (Thromboxane synthase) 與前列環素合成酶 (prostacyclin synthase) 均屬於細胞色素P450超家族的成員。細胞色素P450 是一群含有血紅素基團 (heme) 的酵素，並參與細胞中許多重要分子的氧化與生合成。一般而言，大部分的 P450酵素係藉由催化單氧化反應(monooxygenation) 以執行其生理功能，且反應過程需要NAPH以及氧分子的參與。然而，不同於其他P450酵素，血栓素合成酶與前列環素合成酶具有催化同分異構化反應(isomerization)的活性，由於此兩酵素作用時不需其他cofactors，因而被歸類為第三型細胞色素P450。有趣的是，前列腺素(prostaglandin H2) 為兩者共同的受質，但卻會產生血栓素以及前列環素兩種不同的產物。先前的光譜學研究已指出，兩酵素與受質在立體空間上結合的特異性是控制反應專一性的關鍵。在血栓素合成酶的催化過程中，前列腺素利用O-9與血紅素基團結合；但在前列環素合成酶之血紅素基團則與前列腺素的O-11結合。雖然血栓素合成酶與前列腺素合成酶皆以前列腺素為其生合成的前驅物，兩者卻扮演著相對的生物功能。血栓素能刺激血管收縮及血小板活化凝集；相反的，前列環素則是抑制血管收縮與血小板的活化。因此，此兩生物活性脂共同維繫心血管系統的正常運作，並與動脈粥樣硬化、休克症候群以及癌化過程密切相關。 為了探討前列腺素與酵素結合的立體特異性，我們先前已對前列環素合成酶與抑制物或受質類似物的複合體進行結構解析。結構分析可知活性中心周圍的胺基酸可能影響受質的結合模式並於催化反應扮演著很重要的角色。為了更進一步釐清這些胺基酸的重要性，我們將直接以凡得瓦力和氫鍵與受質作用的W272及N277 分別突變成alanine，希望透過結構的角度了解突變蛋白與受質的交互作用是否發生變化。另一方面，我們也將著手於血栓素合成酶的結構研究，探討與前列腺素結合的立體特異性。 我們已順利判定前列環素合成酶突變蛋白N277A及其與受質類似物複合體的晶體結構，經X-ray 繞射解析度分別為2.37 Å 及 2.57 Å的解析度，且兩者均屬於P212121的空間群。由活性中心的電子密度圖可知，突變後，雖然N277側鏈與受質O-9間的氫鍵消失了，但對受質與血紅素基團結合的方向性以及周圍胺基酸的排列並無太大變化。出乎意料的是，W272A突變會影響N277側鏈於空間中的位置，使其無法與受質形成氫鍵，由於W272的突變蛋白失去催化活性，因此可知W272及N277對催化極為重要。另一方面，為了得到可溶的血栓素合成酶，我們以MAKKTSS親水性序列取代其N端的跨膜區域。然而，目前老鼠血栓素合成酶的純化產量、純度與均質性仍然過低，尚不足以用於結晶，此為未來仍須努力的方向。

Thromboxane synthase (TXAS) and prostacyclin synthase(PGIS) belong to the heme-containing cytochrome P450 superfamily that participates in numerous crucial oxidation processes. However, rather than possessing a monooxygenation activity like most other P450 enzymes, PGIS and TXAS carry out isomerization reactions and require neither NAPH nor O2 for function, they are classified as class III P450. Interestingly, while prostaglandin H2 (PGH2) is a common substrate for both enzymes, thromboxane A2 (TXA2) and prostacyclin (PGI2) are produced by TXAS and PGIS, respectively. Spectroscopic studies indicated that stereospecific substrate binding determines product specificity. In TXAS, the O-9 of PGH2 ligates to the heme-iron, but in PGIS the O-11 of PGH2 serves as the heme ligand. Although TXAS and PGIS catalyze chemical conversion of the same substrate (PGH2), their respective product displays opposite biological activities. TXA2 is a potent stimulator of vasoconstriction and platelet activation. In contrast, PGI2 inhibits vasoconstriction and platelet aggregation. Together, these two bioactive prostanoids mediate key events in atherosclerosis, shock syndromes and carcinogenesis. To understand how the stereospecific PGH2 binding is achieved, our lab has previously determined the crystal structures of PGIS and its complexes with substrate analog and inhibitor. Structural comparison has lead to the identification of residues in the catalytic center that may be crucial for catalysis. To understand these residues catalytic roles in greater detail, residues W272 and N277 have been mutated as alanines, and crystallographic analyses are performed to examine how substrate binding is affected by mutations. In addition, we attempted to initiate structural studies on TXAS to gain more insights on the molecular basis of stereospecific substrate recognition. The crystal structures of ligand-free and substrate analog-bound PGIS-N277A mutant were determined at 2.37 Å and 2.57 Å, respectively. The structure around the enzyme active site reveals that the hydrogen bond between N277 side chain and O-9 of PGH2 was abolished upon mutating N277 to Alanine. However, the spatial orientation of the substrate analog and the arrangement of surrounding residues are not altered. Unexpectedly, the W272A mutation alters the conformation of N277 side chain, leading to the loss of hydrogen bond between N277 and O-9 of PGH2. These observations support the potential importance of W272 and N277 in catalysis and are consistent with the highly conserved nature of these residues. To obtain soluble protein samples for crystallization, TXAS was modified by replacing the N-terminal transmembrane domain with a hydrophilic sequence, MAKKTSS. Nevertheless, homogeneity and yield of purified mouse TXAS remain to be improved before crystallization trials can be performed.