甘藷葉部PI-I胰蛋白?仰制因數活性區及雙元體構造之探討

Abstract

SPLTI (sweet potato leaf trypsin inhibitor)為甘藷葉部經乾旱誘導產生的蛋白質，屬於Potato I family，經過初步分析胺基酸一級排列以及三級結構模擬，比對得到活性區的膠基酸為-R-E-，在Potato I family中較為特殊。SPLTI經由電泳分析得到的分子量為14kDa，為預估分子量的2倍；結構模擬的結果顯示SPLTI並不容易形成分子內雙硫鍵。因而本論文希望能證實-R-E-和TI (trypsin inhibitor)活性的關係以及SPLTI是否以雙硫鍵形成雙元體。於是進一步以定點突變，分析R46M、E47K、C42S和TI活性的關係，R46M與E47K突變蛋白喪失TI活性，C42S仍保有TI活性。接著以β-mercaptoethanol與dithiothreitol處理SPLTI，得到單元體蛋白質。C42S與C59S突變蛋白的分析，證實了雙元體是由此二胺基酸形成的雙硫鍵造成的。此外，也測試了SPLTI以及R46kFE47D雙突變蛋白對於胰凝乳蛋白脢及枯草桿菌蛋白脢的抑制效果，結果SPLTI以及R46M以及R46kFE47D突變蛋白對此二蛋白脢並沒有抑制作用。在酵素-抑制因數複合體的動力學研究，得到SPLTI對於胰蛋白脢的平衡解離常數Ki為0.81±0.24nM; C42S突變蛋白對於胰蛋白脢的Ki=1.73±0.26nM及C59S突變蛋白對於胰蛋白脢的Ki=1.64±0.24nM。最後則對雙元體形成的重要性，與抑制效果的專一性提出合理的解釋。

SPLTI (sweet potato leaf trypsin inhibitor) was drought-induced in sweet potato leaf. The previous study of SPLTI found that it belongs to Potato I family. It is different since the reactive sites of SPLTI might be -R-E- which is obtained by primary sequence alignment and three dimensional (3-D) structural modeling. The molecular weight of SPLTI was l4kDa when analysis by SDSPAGE. It is twice of the molecular weight of 7kDa deduced from amino acid sequence. No apparent disulfide bond was found through molecular modeling analysis. The objectives of the thesis were (1) to demonstrate the relationship of TI (trypsin inhibitor) activity and the reactive site, -R-E-; (2) to study whether the inter-molecular disulfide bonds contribute to dimerization of SPLTI. When using site-directed mutagenesis as the tool, it is proved that R46M and E47K mutated protein lost the TI activity, while C42S did not. The protomer of SPLTI can be separated when treated with β-mercaptoethanol and dithiothreitol, respectively. The contribution of disulfide bond to dimerization of SPLTI was demonstrated by C42S and C59S mutated protein as well. The SPLTI and R46M/E47D double mutated protein did not show inhibitory activity to chymotrypsin and subtilisin. The equilibrium dissociation constant (K1) of SPLTI to trypsin was 0.81±0.24 nM, C42S mutated protein to trypsin was 1.73±0.26nM, and C59S mutated protein to trypsin was 1.64±0.24nM. The importance of dimerization and the specific inhibitory activity of SPLTI were discussed.