台灣龜殼花蛇毒金屬蛋白酶對抑制劑和基質專一性之結構研究

X-ray crystallographic study of a snake venom metalloproteinase from Trimeresurus mucrosquamatus: structural basis for inhibition by endogenous inhibitor and substrate specificity

吳政恆(Cheng-Heng Wu)

指導教授 ： 王惠鈞 ； 孫玉珠

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2007年)

https://doi.org/10.6843/NTHU.2007.00606

摘要

TM-1是從台灣龜殼花分離出來的一種小分子蛇毒蛋白酶，它晶體結構的解析度到1.84Å，經改變後所獲得的R和Rfree 個別是0.179和0.216。TM-1的整體結構是呈現一個扁橢圓形狀，在整個TM-1結構中擁有三個雙硫鍵，分別是在Cys119-Cys198、Cys160-Cys182、Cys162-Cys165的位置上。整個TM-1結構中，裡面有一個zinc ion被四個配體(ligand)所包圍，這四個配體(ligand)包含了三個hisditines和一個水分子，它們形成了tetrahedral geometry的構形。我們發現TM-1和TM-3之間活性位置結構的差異可能顯示了TM-1和TM-3對內生性的抑制劑有不同的敏感性。再者，我們已經藉由塑造的方式把三個內生性的抑制劑(pEKW、pEQW、pENW)放入到TM-1的活性結構位置。我們從抑制劑和TM-1相互作用力的分析結果可以看出抑制劑P-2位置的氨基酸可以和TM-1形成氫鍵和恐水性的作用力，所以抑制劑P-2位置對TM-1的鍵結是很重要的。藉由氧化胰島素B鏈對TM-的蛋白質水解的實驗，我們猜測基質的P-1和P-2位置對TM-1的切割是很重要的。P-1位置所偏好的氨基酸是Leu、Phe和His，而P-2位置所偏好的氨基酸是Leu、Val和Tyr。在這些結論中,我們的結果指示TM-1和TM-3結構的差異可能影響它們對抑制劑的敏感性和基質專一性。除此之外，我們也猜測基質的P-1和P-2位置對TM-1切割基質是重要的。

關鍵字

蛇毒金屬蛋白酶 ； 四面體幾何 ； 內生性抑制劑 ； 相互作用力分析 ； 疏水性作用力 ； 基質專一性

並列摘要

The crystal structure of TM-1, a small snake-venom metalloproteinase (SVMP) isolated from Taiwan habu (Trimeresurus mucrosquamatus), was determined at 1.84 Å resolution with resultant R and Rfree values of 0.179 and 0.216, respectively. The overall structure of TM-1 is an oblate ellipsoid that contains three disulfide crosslinks, Cys119-Cys198, Cys160-Cys182 and Cys162-Cys165. The overall structure of TM-1 contains one zinc ion which is bound to four ligands, including three conserved histidines and one water molecule, displaying a tetrahedral geometry. We find that the distinct active site structures between TM-1 and TM-3 may reflect the different sensitivities toward the endogenous inhibitors from structural comparison of TM-1 and TM-3. Moreover, we have modeled the three endogenous inhibitors, i.e., pyroGlu-Asn-Trp (pENW), pyroGlu-Gln-Trp (pEQW) and pyroGlu-Lys-Trp (pEKW), into the active site of TM-1 structure. Results from interaction analysis of TM-1 and inhibitors show that the P-2 site of inhibitors is important for binding to TM-1 via hydrogen bonds and hydrophobic interactions. By proteolysis experiments of TM-1 using oxidized insulin B-chain as substrate, we sugguest P-1 and P-2 sites of substrates are important for cleavage by TM-1. The preferential amino acids at P-1 site are Leu, Phe and His, while The preferential amino acids at P-2 site are Leu, Val and Tyr. In conclusions, our results indicate that the structural difference between TM-1 and TM-3 may influence their endogenous inhibitor sensitivities and substrate specificity. Additionally, we also suggest that the P-1 and P-2 sites of substrate are critical for cleavage by TM-1.

並列關鍵字

snake-venom metalloproteinase(SVMP) ； tetrahedral geometry ； endogenous inhibitors ； interaction analysis ； hydrophobic interaction ； substrate specificity

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