鏈球菌熱原性外毒素B (Streptococcal pyrogenic exotoxin B ,簡稱SPE B)，它是由化膿性鏈球菌 ( Streptococcus pyogenes ) 所產生的一種細胞外毒素，根據其催化區的分類，將它歸屬於半胱胺酸蛋白酶(cysteine protease)的一種。SPEB 是以40kDa 的酶原 ( Zymogen , ProSPEB ) 形式被鏈球菌分泌至細胞外，之後會經由自動催化 (autocatalysis) 或是經由其他蛋白酶活化形成28 kDa 具有活性之蛋白酶。過去對於SPE B 的研究中指出:在感染宿主時，SPE B 對於化膿性鏈球菌佔有重要的角色，它不僅可以分解細胞間質主要成份 fibronectin 及vitronectin，作用於interleukin-1β (IL-1β) precursor 並產生活化型的 IL-1β而加重發炎反應，誘導吞噬細胞凋亡及降低其吞噬能力等，故SPE B 被認為是化膿性鏈球菌的重要毒性因子而成為極具吸引力的治療標的。研究發現SPE B 的活化中心為半胱胺酸(Cysteine)及組胺酸 (Histidine)，為了深入瞭解SPE B 酵素催化的反應機制，我們利用定點突變的方法構築五株突變株，分別為Q162N (推測與穩定過渡態有關)、W359A (推測位於活化區)、以及W357A、G281A 和V334A (推測與受質結合有關)。另外我們在比對SPE B 胺基酸序列時發現SPE
B 在化膿性鏈球菌中具有多型性。為了瞭解這些多型性的SPE B 在化膿性鏈球菌感染宿主過程中所扮演的角色，我們利用同樣的方法構築T137I、D154N、G308S、G308S/A317S、G384D 五株突變利用大腸桿菌系統表現蛋白及純化，在純化的過程中加入1.5mM 氯化汞以維持其酶原的形式和抑制其活性。在我們的研究中Q162N、W359A 活性和SPE B 相較之下，活性變的非常弱，顯示他們對SPE B 的催化是非常重要的。而Q162N、G281A、V334A 、W357A、W359A 它們切特異性受質C192S 時，發現和原來SPE B 切C192S 的形態不同，顯示它們與受質的結合有關。在多型性方面， 除了G384D 外，其它多型性的SPE B 是影響酵素活性而不改變其受質特異性，詳細的原因到現在還不清楚。而在G384D 切C192S 時，我們發現它的酵素活性降低大約只有SPE B 的二十分之一，且中間產物幾乎都看不見了，和SPE B 切C192S 有八個中間物的結果有顯著的差別。顯示位於C 端loop 上的G384D 也會影響酵素活性和受質特異性，這個結果和我們實驗室在NMR 的結果是符合的。我們依據切割C192S的速率來排列SPE B 及其突變株的相對活性，依序如下: SPEB ~ G308S > T137I > D154N > G308S/A317S > N356D > G384D > W357A > G281A > V334A >W359A > Q162N >> C192S, H340R.。綜合以上結果顯示SPE B 有六個區域：Y160-G163，T190-A196，S280-S286，G323-F342，W357-W359，和 A376-A391在催化活性和受質結合扮演重要角色，也讓我們了解SPE B 的分子作用機制和其多型性對功能上的影響。

　　Streptococcal pyrogenic exotoxin B (SPE B) is an extracellular cysteine protease which is secreted by Streptococcus pyogenes. SPE B is initially expressed as a 40kDa zymogen and subsequently converted to a 28 kDa active protease by autocatalysis or proteolysis. SPEB participates in the dissemination, colonization, and invasion of bacteria and the inhibition of wound healing. Many reports on SPEB infection reveal that the spe B gene is extremely polymorphic among strains of S. pyogenes. However, little is known about the relationships between the genetic variation of spe B and the diseases made by S. pyogenes. In order to investigate the polymorphism and enzyme mechanism, SPEB and its mutants werw expressed in E. coli system and purified to homogeneity. The Q162, G281/V334/W357, and W359 residues were involved in stabilization state, substrate binding, and active site, respectively. The mutants of Q162N, G281A, V334A, W357A, and W359A almost lost their protease activity, implicating that these residues are essential for the catalytic activity. The polymorphic mutants T137I, D154N, G308S, and G308S/A317S have little effects on their protease activity. In contrast, the polymorphic mutation of G384 to D in the C-terminal loop caused a 20-fold decrease in activity. Our autocatalysis analysis showed that this low activity was due to a slow intermolecular processing of zymogen maturation. This result is consistent with our NMR data that the G384 residue plays an important role in substrate binding. The relative protease activity of these mutants exhibit in the following order: SPEB ~ G308S > T137I > D154N > G308S/A317S > N356D > G384D > W357A > G281A > V334A > W359A > Q162N >> C192S, H340R. Taken together, residues of six regions, including Y160-G163, T190-A196, S280-S286, G323-F342, W357-W359, and A376-A391, of SPE B may play important roles in both catalytic catalysis and substrate binding. This study will extend our understanding of the molecular mechanism and the polymorphic effect of SPE B on S. pyogenes infection.

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