以法呢焦磷酸酯螢光類似物探測十一異戊二烯焦磷酸合成酶的活性中心和反應構形變化

Probing the Active Site and Conformational Change of Undecaprenyl Pyrophosphate Synthase by Farnesyl Pyrophosphate Fluorescent Analogues

李育瑾(Yu Chin Li)

指導教授 ： 陳昭岑

國立臺灣大學/理學院/化學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.10042

摘要

大腸桿菌 (E. Coli) 的十一異戊二烯焦磷酸合成酶 (UPPs ，undecaprenyl pyrophosphate synthase) 能催化連續的縮合反應，將一個法呢焦磷酸酯 (FPP，farnesyl pyrophosphate) 與八個異戊二烯焦磷酸酯 (IPP，isopentenyl pyrophosphate) 縮合成一個C55 UPP。UPP為大腸桿菌lipid Ⅱ的載體，可將之翻轉出細胞膜外繼而繼續進行合成細胞壁。
我們成左漲X成出四個帶有螢光基團的FPP螢光類似物：14 (NBD-FPP)、24 (fluorescein-FPP)、34 (MANT-ESTER-FPP)、44 (MANT-AMIDE-FPP)。利用穩定狀態螢光光譜儀得到四種類似物自身的光學特性，並利用其光學特性觀察與UPPs結合的情況，及利用螢光滴定實驗求得類似物與UPPs的解離常數Kd；另外也利用了酵素動力學實驗，求出四種類似物對UPPs的抑制常數Ki和半抑制濃度IC50。
結果化合物24的抑制能力最好，且因為與UPPs結合前後螢光強度改變，所以可利用其特性在生物體內細胞的作造影成像。化合物34能成為不錯的FPP替代物，且因為Kd夠小，可利用來作為UPPs抑制藥物的篩選。而化合物14、24、34、44都將可能發展成為研究各種不同FPP-binding酵素的工具和作為高速篩選的探針。

關鍵字

法呢焦磷酸酯螢光類似物 ； 十一異戊二烯焦磷酸合成酶

並列摘要

Undecaprenyl pyrophosphate synthase (UPPs) of E. Coli. catalyzes the condensation of eight molecules of isopentyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate C55 undecaprenyl pyrophosphate, which serves as a carrier in transporting lipid II across the membrane in bacterial cell wall biosythesis.
We successively synthesized four FPP fluorescent analogues: 14 (NBD-FPP), 24 (fluorescein-FPP), 34 (MANT-ESTER-FPP) and 44 (MANT-AMIDE-FPP). We obtained spectroscopic characterization of four analogues by steady-state fluorescence spectrophotometer, and we utilized those to observe the ligand interactions with UPPs. And we also got different kinetic parameters of four analogues. 24 has the best competitive inhibition, and when binding with UPPs, the difference of fluorescent intensity can be seen by eyes. 34 can serve as alternative substrate for UPPs, and could be used as drug screening. Analogues 14, 24, 34, 44 could be used to study different FPP-binding proteins and served as high throughput probes.

並列關鍵字

Farnesyl Pyrophosphate Fluorescent Analogues ； Undecaprenyl Pyrophosphate Synthase

參考文獻

1. Poulter, C. D.; Rilling, H. C. Biosynthesis of Isoprenoid Compounds (Spurgeon, S.L., ed.) John Wiley & Sons, New York. 1981.

2. Poulter, C. D. J. Agric. Food Chem. 1974, 22, 167-173.

3. Sinensky, M. Biochim. Biophys. Acta. 2002, 1484, 93-106.

7. Robyt, J. Essentials of Carbohydrate Chemistry. Springer-Verlag, New York. 1998.

8. Casey, P. J. Biochem. Soc. Trans. 1995, 23, 161-166.