合成法呢基焦磷酸和異戊二烯焦磷酸類似物來探討順式及反式-異戊二烯轉移酵素的催化機制

Abstract

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.