探討α-L-arabinofuranosidase E223單點突變造成的反應機制之改變

Abstract

木腐黴菌（Trichoderma koningii G-39）阿拉伯呋喃糖苷酵素(ABF)，屬於醣類水解酵素第54家族，其催化機制為保留機制（retention）且速率決定步驟為去醣基化（dearabinosylation）。其中E223和D299分別扮演親核基和一般酸/鹼基團之角色。在本研究中我們利用溶劑同位素效應（isotope solvent effect）再次強化D299在催化反應中之角色。然而，有趣的是突變酵素（E223G）仍保有明顯的活性。Aspergillus kawachii ABF是目前此家族中唯有三維立體結構，利用蛋白質序列相比對，發現兩者之相同度（identity）達72%，且其中活性區內之重要胺基酸均高度保留。因此我們利用其Aspergillus kawachii ABF之三維立體結構為模板，進行T. koningii ABF結構模擬。發現D191的位置可取代E223成為另一個重要胺基酸殘基，其與受質異位性碳原子（anomeric carbon）距離約4 Å，故當E223被突變成G223時，可能使E223G反應轉變為反轉機制（inverting），而本研究旨於探討此突變酵素之催化性質與機構。 本研究利用Pichia pastoris系統表現酵素，經由單菌落PCR確認基因轉殖結果，再由活性測試與蛋白質電泳方法，可以成功的篩選表現效率最佳之單菌落，其再進一步被誘導產生大量酵素。經80%飽和度之銨鹽沉澱與陽離子交換樹脂管柱層析，可以得到均質度達95%的酵素以利動力學研究使用。 根據酵素動力學的研究E223G/D191N與E223G/D191G活性值（kcat/ Km）降為E223G的0.69% ~ 4.6%，顯示D191在E223G中可能是重要胺基酸。另外，E223G/D299N活性值降為wild type的0.12%，E223G的2.8%，這很有可能說明D299不僅在wild type之中，同時也在E223G中是重要胺基酸。 E223G 和Wild type的pH activity profile顯示兩鐘形曲線分佈，這兩酵素催化過程都分別由兩重要胺基酸基團調控。對其他突變酵素之pH profile研究顯示，其中E223G/D191G之 pKa2=5.56，並且沒有pKa1。E223G/D299N之pKa1=2.23沒有pKa2。這些證據都顯示D191和D299分別是E223G的一般鹼基團與一般酸基團。 由分子模擬得知，D299和E223距離約為6 Å，是預期中保留機制兩重要胺基酸残基的距離。D299與D191距離為7.5 Å，則是預期可進行反轉機制的距離，因此我們認為E223G之催化反應為構型反轉之機構，而D191與D299是其重要胺基酸殘基，分別扮演一般酸與一般鹼之重要角色。

Convert a retaining □-L-arabinofuranosidase to inverting enzyme by single point mutation on E223

Student：Kai-Yu Chen Advisor：Dr. Yaw-Kuen Li

Department of Applied Chemistry National Chiao-Tung University

ABSTRACT The □-L-arabinofuranosidase (ABF) from Trichoderma koningii G-39 is a retaining enzyme belonging to GH family 54. Our previous study showed that breakdown of arabinosyl-enzyme intermediate is the rate limiting step of the catalytic reaction. The essential groups are E223 (nucleophile) and D299 (general acid/base). In this study, the investigation of kinetic solvent isotope effect re-confirmed that the D299 functions as the general acid/base in the catalytic reaction. Surprisingly, E223G mutant was found to remain significant activity, while E223Q was completely inactive. The structure of T. koningii ABF was obtained from the homology simulation by using the structure of Aspergillus kawachii ABF (family 54) as the template. The structure exhibited that the distance between D191 and anomeric carbon of substrate is about 4 Å and the space is suitable for accommodating a water molecule. D191 was, thus, proposed to be essential for the catalysis of E223G, and the mechanism of E223G might become an inverting process. For studying this hypothesis, a series of double mutants, such as E223G/D191N, E223G/D191G and E223G/D299N, were constructed and over-expressed in Pichia pastoris system. The colonies with high-level expression were selected through various steps of validation including colony PCR to confirm gene insertion and activity assay or protein electrophoresis (SDS-PAGE) to evaluate the protein expression level. After 80% ammonium sulfate precipitation following by a cation-exchanged chromatographic separation, enzymes can be purified to reach 95% homogeneity and used for further study. Kinetic study revealed that the relative activity (kcat/Km) of E223G/D191N, E223G/D191G and E223G/D299N are 0.69%~4.6% of that of E223G. A bell-shaped pH-profile of E223G showed the catalytic activity of this enzyme is mediated by two pKas, 1.8 and 4.2. However, a sigmoidal pH-profile was observed for E223G/D191G (pKa2= 5.56) and for E223G/D299N (pKa2= 2.23), indicating that D191 and D299 are the general base and the general acid of E223G, respectively. These findings are consistent with the suggestions obtained from the simulated structure of T. koningii ABF.