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Probing the Catalytic Mechanisms of Cis-type and Trans-type Prenyltransferases Using the Synthesized Analogs of FPP and IPP
No full text四異戊二烯焦磷酸合成酵素 (Geranylgeranyl pryophsophate synthase, GGPPs)、八異戊二烯焦磷酸合成酵素 (Octaprenyl pryophsophate synthase, OPPs) 、十一異戊二烯焦磷酸合成酵素 (Undecaprenyl pryophsophate synthase, UPPs) 都是屬於異戊二烯轉移酵素 (prenyltransferase)。不同種類的酵素會催化法呢基焦磷酸 (Farnesyl diphosphate, FPP) 與不同數目的異戊二烯焦磷酸 (Isopentenyl diphosphate, IPP) 進行1’-4縮合反應 (1’-4 condensation reaction) 形成碳鏈長短不同的產物。根據法呢基焦磷酸與異戊二烯焦磷酸縮何反應 (condensation reaction) 後產生的雙鍵立體化學 (stereochemistry) 結構不同，異戊二烯轉移酵素可以分成反式 (trans-type) 與順式 (cis-type) 兩種。四異戊二烯焦磷酸合成酵素 (GGPPs) 與八異戊二烯焦磷酸合成酵素 (OPPs)為反式-酵素，而十一異戊二烯焦磷酸合成酵素 (UPPs) 則為順式-酵素。對於1’-4縮合反應 (1’-4 condensation reaction) 在過去曾經有兩種反應機制被提出 (1) 離子化-縮合-消去 (ionization- condensation-elimination mechanism)，(2) 縮合-消去 (condensation-elimination mechanism)。
在本篇研究中我們分別合成呢基焦磷酸與異戊二烯焦磷酸的類似物，並且利用它們來研究這三種酵素的催化機制，尤其是特別針對順式-十一異戊二烯焦磷酸合成酵素。以強拉電子基團溴原子取代甲基的異戊二烯焦磷酸類似物 (3-bromo-3-butenyl diphosphate)，實驗中它能夠有效減慢縮合反應的發生，並且在鹼性環境下我們能夠在四異戊二烯焦磷酸合成酵素與八異戊二烯焦磷酸合成酵素的反應裡成功萃取到反應的中間物法呢醇 (farnesol)，這個結果讓我們認為反式-異戊二烯轉移酵素的反應過程是經由離子化-縮合-消去反應機制。以[1-14C]同位素法呢基焦磷酸取代[1-12C]同位素法呢基焦磷酸來進行實驗，發現酵素反應中動力學同位素效應 (kinetic isotope effect) 對於異戊二烯焦磷酸的消耗，在十一異戊二烯焦磷酸合成酵素的反應裡 (1.14 ± 0.04) 小於八異戊二烯焦磷酸合成酵素的反應 (1.61 ± 0.07) 及四異戊二烯焦磷酸合成酵素的反應 (1.73 ± 0.07)。總結實驗結果，針對十一異戊二烯焦磷酸合成酵素的反應，因為不同同位素的法呢基焦磷酸對於異戊二烯焦磷酸的消耗沒有明顯的動力學同位素效應，且無法萃取到反應的中間物法呢醇，讓我们認為順式-十一異戊二烯焦磷酸合成酵素的反應過程是經由縮合-消去的反應機制。Geranylgeranyl pryophsophate synthase (GGPPs, C20), octaprenyl pyrophosphate synthase (OPPs, C40), and undecaprenyl pyrophosphate synthases (UPPs, C55) are prenyltransferases which catalyze chain elongation of farnesyl diphosphate (FPP) via 1’-4 condensation reaction with various numbers of isopentenyl pyrophosphate (IPP) units to generate different isoprenoids. Based on the stereochemistry of the double bond formed during IPP condensation, prenyltransferases are classified into trans- and cis-types. GGPPs and OPPs are trans-type prenyltransferase, while UPPs belongs to the cis-type. The possible mechanisms for 1’-4 condensation reaction that have been proposed are (1) ionization-condensation-elimination mechanism (sequential mechanism), and (2) condensation-elimination mechanism (concerted mechanism).
In this study, we synthesized analogs of FPP and IPP to probe the reaction mechanisms of these three prenyltransferases, particularly that of the cis-type UPPs. By substituting the methyl group at C3 position of IPP with an electron-withdrawing bromo group, the resulting analog of IPP, 3-bromo-3-butenyl diphosphate, slowed down the rate of the condensation reaction. Trapping of the farnesol intermediates in the catalytic active site of GGPPs and OPPs using radiolabeled FPP led us to propose that the reaction mechanisms of trans-type prenyltransferases go through an ionization- condensation-elimination mechanism. Using [1-14C] FPP as the substrate in place of [1-12C] FPP, the kinetic isotope effect on the consumption of IPP for UPPs reaction (1.14 ± 0.04 ) was smaller than those for OPPs reaction (1.61 ± 0.07) and GGPPs reaction (1.73 ± 0.07). The lack of apparent isotope effect and the fact that no farnesol intermediate was trapped, indicating that the cis-type UPPs reaction may proceed through a concerted condensation-elimination mechanism.中文摘要 1
ABSTRACT 3
ABBREVIATION 6
(1) INTRODUCTION
1-1 Isoprenoid 7
1-2 Classification of Prenyltransferases 7
1-3 Isoprenyl Pyrophosphate Synthases 8
1-4 Trans-type Prenyltransferases: Geranylgeranyl Diphosphate and Octaprenyl Diphosphate Synthase 9
1-5 Cis-type Prenyltransferases: Undecaprenyl Pyrophosphate Synthase 10
1-6 Specific Aims of This Study 11
(2) MATERIAL AND METHODS
2-1 Chemicals 14
2-2 Synthesis of Analogs of Farnesyl Diphosphate and Isopentenyl Diphosph
ate 14
2-2-1 Synthesis of 13-Trifluorofarnesyl Diphosphate 14
2-2-2 Synthesis of 3-Bromo-3-butenyl Diphosphate 15
2-3 Expression and Purification of Prenyltransferases 16
2-3-1 Purification of His-tagged UPPs and Removal of Tag 16
2-3-2 Purification of His-tagged OPPs 17
2-3-3 Purification of His-tagged GGPPs and Removal of Tag 17
2-4 Trapping Farnesyl Cation Intermediate in the Prenyltransferase Reactions 18
2-4-1 Trapping Farnesyl Cation Intermediate during the Prenyltransferase Reactions in the presence of [3H] FPP 18
2-4-2 Analysis of Farnesyl Cation Intermediate for the GGPPs Reaction 19
2-4-3 Trapping Farnesyl Cation Intermediate Using [3H] FPP and Br-IPP as Substrates 19
2-4-4 Analysis of Farnesyl Cation Intermediate by TLC 20
2-5 Measurements of Kinetic Constant 21
2-5-1 Kinetic Measurements of 13-Trifluorofarnesyl Diphosphate 21
2-5-2 Measurements of Inhibition Constants of 13-Trifluorofarnesyl Diphosphate 21
2-6 Isotope Effect Experiments 22
(3) RESULTS
3-1 Probing the Prenyltransferase Reaction Mechanism Using Fluoro- Substrate Analog 24
3-1-1 Synthesis of 13-Trifluorofarnesyl Diphosphate 24
3-1-2 Use of 13-Trifluorofarnesyl Diphosphate as Substrate to Replace FPP in Prenyltransferase Reaction 24
3-1-3 Synthesis of 3-Bromo-3-butenyl Diphosphate 25
3-1-4 Use of 3-bromo-3-butenyl Diphosphate as Substrate to Replace IPP in Prenyltransferase Reactions 25
3-2 Trapping Farnesyl Cation Intermediate as a Direct Proof of the Reaction Mechanism 26
3-2-1 Trapping Intermediate with [14C] FPP 26
3-2-2 Trapping Intermediate in the Presence of [14C] FPP and Br-IP 27
3-3 Isoptope Effect for UPPs Reaction Probed by [14C] FPP 27
3-4 A Different Mechanism for UPPs Reaction 28
3-5 Substrate Analog Binds Poorly in UPPs Active Site 29
(4) DISCUSSION
4-1 Catalytic Mechanism of Cis-type UPPs 30
4-2 Catalytic Mechanism of Trans-type GGPPs and OPPs 31
(5) REFERENCE 33
(6) TABLE 40
(7) SCHEME 41-42
(8) FIGURE 43-60
(9) APPENDIX 61-8
Mechanism of cis-prenyltransferase reaction probed by substrate analogues
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