順式─十一異戊二烯焦磷酸及反式─八異戊二烯焦磷酸合成酶催化的重要胺基酸及其反應機制

Essential amino acids involved in the catalysis of cis-type undecaprenyl pyrophosphate synthase and trans-type octaprenyl pyrophosphate synthase from E. coli and their reaction mechanisms

黎世俊(Shih-Chun Li)

指導教授 ： 梁博煌

國立臺灣大學/生命科學院/生化科學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.02202

摘要

類異戊二烯族化合物廣泛分布於自然界是由異戊二烯焦磷酸構成的聚合物。此類化合物的生合成是由一類異戊二烯轉移酵素所催化。這類酵素催化多個含5個碳異戊二烯焦磷酸和含15個碳法呢基焦磷酸結合生成長鏈產物。這些酵素扮演著重要的生理弁遄A例如十一異戊二烯焦磷酸合成酵素產生的55個碳的產物可攜帶醣質以合成細菌胞壁，此酵素的活性抑制劑可作為抗生素藥物；八異戊二烯焦磷酸合成酵素(Octaprenyl pyrophosphate synthase)催化五個異戊二烯焦磷酸和法呢基焦磷酸反應產生含40個碳的產物可作為ubiquinone支鏈。我們先前已研究此二酵素的反應動力學及三度空間結構，根據結構、一些位於活性區域的氨基酸可能參與基質結合或催化反應，尤其是反應中扮演general acid and base的氨基酸仍不清楚，本篇論文我企圖針對酵素活性對於酸鹼值的變化配合定點突變來找出這些重要的氨基酸，也發展一些新穎的研究方法。

關鍵字

類異戊二烯 ； 轉移酵素 ； 合成 ； 焦磷酸

並列摘要

Isoprenoids are widely distributed natural polymers containing isopentenyl pyrophosphate (IPP) as building block. The biosyntheses of isoprenoids are carried out by a group of enzymes called prenyltransferases which catalyze the head to tail condensation between IPP and farnesyl pyrophosphate (FPP) to make linear long-chain products. These FPP-utilizing enzymes play essential biological role, e.g. the C55 undecaprenyl pyrophosphate (UPP) generated by UPP synthase (UPPs) serves as a lipid carrier to transport lipid II across the bacterial cell membrane for peptidoglycan synthesis of the cell wall; 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. Previously, we have solved the complete kinetic pathways and 3-D structures of E. coli UPPs and OPPs. Based on the crystal structure, some amino acids in neighborhood of the active site are proposed to play important roles in substrate binding and the reaction. However, the amino acids which act as general acid and base in the catalysis have not been identified. In this study, we utilized pH profile in conjunction with site-directed mutagenesis to examine the important residues. Some novel method of study has also been developed.

並列關鍵字

UPPs ； prenyltransferases ； farnesyl pyrophosphate ； OPPs ； isopentenyl pyrophosphate ； Isoprenoids

參考文獻

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