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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 梁博煌(Po-Huang Liang) | |
| dc.contributor.author | Keng-Ming Chang | en |
| dc.contributor.author | 張耕銘 | zh_TW |
| dc.date.accessioned | 2021-06-13T07:07:46Z | - |
| dc.date.available | 2010-07-30 | |
| dc.date.copyright | 2005-07-30 | |
| dc.date.issued | 2005 | |
| dc.date.submitted | 2005-07-26 | |
| dc.identifier.citation | REFERENCES
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35742 | - |
| dc.description.abstract | 中文摘要
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(isopentenyl pyrophosphate, IPP)和一個法呢基焦磷酸(Farnesyl pyrophosphate, FPP)反應產生含40個碳的產物可作為對ATP生合成重要元素ubiquinone或menaquinone支鏈。此類酵素具有二段含Asp的DDXXD序列，其可和鎂離子相結合來結合酵素基質的焦磷酸，但根據我們先前已研究且解出三度空間結構，二個硫酸根離子分別結合於第一個DDXXD序列及一個帶正電荷的所在，我們因此決定用定點突變來研究受質結合區，在可能結合受質的第一個DDXXD序列包括D84, D85, D88及R93突變成Ala，結果發現這些氨基酸和FPP的結合較相關，而帶正電荷部位包括K45, R48, H77, 及R94和IPP的結合較相關。 這些結論也由突變酵素的結晶結構所支持。我們又使用螢光方法來決定受質及產物和酵素的結合，並發現酵素和受質的結合沒有先後順序且產物的離去為速率決定部驟。同時我也研究酵素活性對於酸鹼值的變化，發現H77可能是扮演general base的角色。另外我還研究受質的專一性，金屬離子的功能，並捕捉到反應中間物以及利用電腦模擬抑制劑等。 | zh_TW |
| dc.description.abstract | ABSTRACT
Octaprenyl pyrophosphate synthase (OPPS) catalyzes consecutive condensation reactions of one molecule of farnesyl pyrophosphate (FPP) with five molecules of isopentenyl pyrophosphates (IPP) to generate C40 octaprenyl pyrophosphate (OPP). The octaprenyl group constitutes the side chain of ubiquinone or menaquinone, an essential component for ATP synthesis. Two DDXXD motifs conserved among all the trans-prenyltransferases were suspected to bind FPP and IPP substrates via Mg2+. However, two sulfate ions S1 and S2 were found binding in the first DDXXD and a positively-charged pocket in the active site of T. maritima OPPS, respectively. To examine the substrate-binding mode, we built a structural model for E. coli OPPS based on its sequence homology to the T. maritima OPPS and performed site-directed mutagenesis. The amino acids D84, D85, and D88 of the first DDXXD and R93, which presumably bind the first sulfate ion, are essential for FPP binding and catalysis. The K45, R48, H77, and R94, which presumably bind the other sulfate ion, are more important for IPP binding and catalysis. These results suggest a substrate-binding mode, in which FPP binds to the first DDXXD motif and IPP binds to the S2 site. This conclusion is also supported by the crystal structures of the mutant T. maritima OPPS. By using fluorescent stopped-flow method, the kinetic scheme for the OPPS reaction was determined. OPPS binds FPP and IPP in a random order and the product release represents the rate limiting step. The trapping of the reaction intermediate, pH profile support the ionization-condensation-elimination mechanism for OPPS reaction. We also determined the pKa of essential amino acids derived from pH profile studies of wild type and H77D and H77Q mutants and found that H77 might act as a general base in the OPPS reaction. Moreover, the role of metal ion in catalysis and the substrate specificity, as well as the computer modeling of the inhibitors were also studied. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T07:07:46Z (GMT). No. of bitstreams: 1 ntu-94-R92b46034-1.pdf: 1671328 bytes, checksum: 3769a06be30f61e9656eb7d342e793a2 (MD5) Previous issue date: 2005 | en |
| dc.description.tableofcontents | TABLE OF CONTENTS
ABSTRACT………………………………………………………………………….. 4 ABBREVIATIONS……………………………………………………………………7 INTRODUCTION ………...…………………………………………………………..8 EXPERIMENTAL PROCEDURES……...…………………………………………..14 RESULTS ………...………………………………………………………………….27 DISCUSSION ………..……………………………………………………………...38 TABLES ………..……………………………………………………………………46 Table 1A. Primers used to construct E. coli OPPS mutants in this study………46 Table 1B. Primers used to construct T. maritima OPPS mutants in this study…47 Table 2. The kinetic parameters of E. coli OPPS active site mutants…………..48 Table 3. Characterization of OPPS with different allylic substrates……………49 Table 4. The activities of OPPS with different metal ions……………………...49 Table 5. The role of pyrophosphate in allylic substrates………………………..49 FIGURES …………………………………………………………………………....50 Figure 1. Distribution of prenyl pyrophosphate………………………………...50 Figure 2. Reaction of OPPS and chemical structure of ubiquinone…………….51 Figure 3. Alignment of amino acid sequences of the T. M. OPPS, E. coli OPPS, Mucor SPPS, fission yeast DPPS, Thermoplasma GGPPS, and Thermoplasma FPPS…………………………………………………..52 Figure 4. The strucutre of T. maritima OPPS…………………………………...54 Figure 5. Alignment of amino acid sequences of the E. coli OPPS, T. M. OPPS, E. coli FPPS, and avian FPPS…………………………………………….56 Figure 6. (A) Superimposition of crystal structure of T.M. OPPS and the computer modeling structure of E. coli OPPS. And active site structure of E. coli OPPS………………………………………………………...57 Figure 7. The active site crystal structures of K41A, R44A, H74A, R90A and R91A T.M. OPPS mutants……………………………………………..59 Figure 8. The molecular ruler mechanism of product chain length determination for T.M. and E. coli OPPS……………………………………………..61 Figure 9. Products synthesized by wild-type, A79Y, M123A, M135A, and M123A/M135A OPPS…………………………………………………62 Figure 10. Superimposition of the bound substrates and sulfate ions found in T.M. OPPS, E. coli FPPS, avian FPPS in the active site of E. coli OPPS…..64 Figure 11. (A) The fluorescence spectra and stopped-flow trace of OPPS and 5 | |
| dc.language.iso | en | |
| dc.subject | 反式─八異戊二烯焦磷酸合成酶 | zh_TW |
| dc.subject | trans-type octaprenyl pyrophosphate synthase | en |
| dc.title | 反式─八異戊二烯焦磷酸合成酶催化的反應機制 | zh_TW |
| dc.title | Reaction mechanism of trans-type octaprenyl pyrophosphate synthase from E. coli and Thermotoga maritima | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 93-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 邱式鴻,林俊宏 | |
| dc.subject.keyword | 反式─八異戊二烯焦磷酸合成酶, | zh_TW |
| dc.subject.keyword | trans-type octaprenyl pyrophosphate synthase, | en |
| dc.relation.page | 84 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2005-07-27 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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