第一部份：鱟血清中細菌結合蛋白表達及特性研究 第二部份：大腸桿菌及耐熱菌之八異戊二磷酸合成酵素之反應機制及動力學研究

Abstract

第一部份：鱟血清外源凝集素-1和-2在天賦免疫系統抵禦上，扮演和細菌結合的功能。先前劉德勇教授實驗室從台灣鱟血清中分離出二種外源凝集素[Chiou, S-T., Chen, Y.-W., Chen, S.-C., Chao, C.-F. and Liu, T.-Y.(2000) J. Biol. Chem.275,1630-1634]，及克隆它們的基因發現其為分子量26-kDa 和12-Da的醣蛋白[Chen, S.-C., Yen, C.-H., and Liu, T.-Y,(2001)J.Biol.Chem.276,9631-9639]。描述在本論文中，我使用酵母菌表達系統使這些蛋白分泌至培養基而能簡易純化及鑑定重組蛋白特性。表達之蛋白由於不同程度的醣化而在SDS-PAGE上出現二個band，其可被切醣酵素切成和不具醣化的突變蛋白在SDS-PAGE上相同位置。使用ELISA研究其ligand結合專一性顯示鱟血清外源凝集素-1和N-acetyl醣類結合而鱟血清外源凝集素-2辨識細菌表面脂多醣之O-antigen部份。當外源凝集素-1的醣化序列的Asn被Asp所取代，即失去細菌結合能力表示醣化對鱟血清外源凝集素-1是必需的。另一方面、外源凝集素-2醣化序列的N3D突變並不影響其功能。就蛋白組成而言、分子間雙硫鍵所形成的dimer對於鱟血清外源凝集素-2是必需的且脂多醣造成其多元體形成。 第二部份：八異戊二烯焦磷酸合成酵素(OPPs)催化五個異戊二烯焦磷酸(IPP)和法呢基焦磷酸(FPP)反應生成產物八異戊二烯焦磷酸，其可形成ubiquinone的支鏈。由於長鏈產物離開酵素不易、此類酵素常需要加入detergent或其他因數維持其最佳活性。先前發現加Triton可將十一異戊二烯焦磷酸合成酵素的速率決定部驟從產物離開轉變成異戊二烯焦磷酸化學反應[Pan, J.J., Chiou, S.T., and Liang, R.H.(2000) Biochemistry 39,10936-109421]。為瞭解八異戊二烯焦磷酸合成酵素之活性激發機制，我們測量酵素單一循環反應(single-turnover)發現異戊二烯焦磷酸的化學反應速率常數為2 s-1、100倍大於steady stat測得的反應速率常數0.02 s-1, 細菌細胞內高分子量蛋白和Triton皆可將反應速率常數增加約三倍、但不足以造成100倍效果。產物以burst方式形成亦證明在有或無Triton狀況，產物離開很慢為速率決定部驟。此部份已發表[Pan, J.J., Kuo, T.H., Chen, Y.K., Yang, L.W., and Liang, P. H. (2002) Insight into the activation mechanism of E. coli octaprenyl pyrophosphates synthase derived from pre-steady-state kinetic analysis. Biochim. Biophys. Acta 1594, 64-73]. 我亦表達、純化及進行動力學研究對耐熱菌的八異戊二烯焦磷酸合成酵素。此酵素在室溫下的活性為0.005 s-l，且活性隨溫度昇高而呈exponential增加。和大腸桿菌不同的是耐熱菌的八異戊二烯焦磷酸合成酵素的化學反應為速率決定部驟。反應產物為40個碳的八異戊二烯焦磷酸且其轉變成45個碳速率幾乎可以忽略。在有10 ?M OPPs-FPP複合物和1?M IPP單一循環反應(single-turnover)狀況下，只有C20而非由大腸桿菌酵素所產生的C20-C40。這些數據顯示大腸桿菌和耐熱菌的八異戊二烯焦磷酸合成酵素動力學的差異，反應出耐熱菌酵素較高的活性區域穩定度。

PartⅠ: Tachypleus Plasma Lectin-1 and -2 (TPL-1 and -2), which bind bacteria for innate host defense, were previously isolated from the hemolymph of Taiwanese Tachypleus tridentatus [Chiou, S.-T., Chen, Y.-W., Chen, S. -C., Chao, C.-F. and Liu, T.-Y.(2000)J.Biol.Chem.275,1630-1634] and their genes encoding a 26-kDa and a 12-kDa glycol-proteins were cloned in Dr. Liu's laboratory [Chen, S. -C., Yen, C. -H., and Liu, T .-Y.,(2001)J.Biol.Chem.276,9631-9639]. In the present study, TPL-1 and -2 produced using yeast and the recombinant proteins secreted into medium were purified and characterized. The purified lectins show two bands on the SDS-PAGE as resulted from heterogeneous glycosylation and the endo-H glycosidase treatment could generate a single band, which migrates on the SDS-PAGE at the same position as non-glycosylated mutant protein. Ligand specificity examination using ELISA assay reveals that TPL-1 interacts with N-acetyl saccharides and TPL-2 recognizes O-antigen of bacterial lipopolysaccharides. The glyco moiety of the TPL-1 is essential for its function as the substitution of Asn in the N-glycosylation site with Asp abolishes the ligand binding affinity. On the other hand, N3D mutant TPL-2 retains bacterial binding activity. The dimer formation by intermolecular disulfide linkage is essential for TPL-2 activity and the LPS induces its oligomerization. PartⅡ.Octaprenyl pyrophosphate synthase (OPPs) catalyzes the sequential condensation of five molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate (FPP) to generate all-trans C40-octaprenyl pyrophosphate, which constitutes the side chain of ubiquinone. Due to the slow product release, a long-chain polyprenyl pyrophosphate synthase often requires detergent or other factor for optimal activity. Our previous studies in examining the activity enhancement of E. coli undecaprenyl pyrophosphate synthase have demonstrated a switch of rate determining step from product release to IPP condensation reaction in the presence of Triton [Pan, J.J., Chiou, S.T., and Liang, P.H.(2000) Biochemistry 39,10936-109421]. In order to understand the mechanism of enzyme activation for E.coli OPPs, a single-turnover reaction was performed and the measured IPP condensation rate (2 s-l) was 100 times larger than the stead-state rate (0.02 s-1). The high molecular weight fractions and Triton could accelerate the steady-stat rate by 3-fold (0.06 s-1) but insufficient to cause full activation (100-fold). A burst product formation was observed in enzyme multiple turnovers indicating a slow product release. This part has been published [Pan, J.J., Kuo, T.H., Chen, Y.K., Nhng, L. W., and Liang, P.H. (2002) Insight into the activation mechanism of E. coli octaprenyl proophosphate synthase derived from pre-steady-state kinetic analysis. Biochim. Biophys. Acta 1594,64-73]. Moreover, a putative gene encoding for OPPs from T. maritima, an anaerobic and thermophilic bacterium,was expressed, purified and its kinetic pathway was determined. The enzyme activity at 25℃ is 0.005 s-1 under steady-state condition and exponentially increased with elevated temperature. In contrast to E. coli OPPs, IPP condensation rather than product release is rate limiting in enzyme reaction. The product of chain elongation catalyzed by T maritima OPPs is C40 and the rate of its conversion to C45 is negligible. Under single-turnover condition with 10 ?M OPPs. FPP complex and 1 pM IPP, only the C20 was formed rather than C20-C40 observed for E.coli enzyme. These data reveal the differences in kinetic properties of OPPs from T maritima and E.coli, reminiscent of lower enzyme activity at room temperature, higher product specificity, higher thermal stability and lower structure flexibility for the thermophilic enzyllle.