利用定點飽和突變的方式對於氧化鯊烯環化酵素內 假設活性區域殘基進行結構與反應關係之研究

Abstract

在動物、真菌以及高等植物中的氧化鲨烯環化酵素會催化氧化鲨烯形成四環或是五環的三萜類化合物。由於這種酵素只需要一個步驟就可以完成複雜的環化以及重排反應，近半世紀以來引起科學家極大的興趣。 在1992年，Griffin提出芳香族的胺基酸可以利用其π電子來穩定中間產物所產生的碳陽離子，故芳香族胺基酸為重要殘基的可能扮演著十分重要的角色。 為了釐清D環上的擴環反應是由哪些高度保留性的胺基酸所影響，我們進行了定點飽和突變，特別是針對Pisum sativum鲨烯環化酵素中的 Phe125以及Saccharomyces cerevisiae鲨烯環化酵素中的Met105進行實驗。 在Saccharomyces cerevisiae M105定點突變實驗中，在突變成G/A/S/N/Q/K會產生protosta-20,24-dien-3β-ol，藉由這結果的產生，推測M105所扮演的角色是影響重排反應以及脫氫反應的位置，但是並未影響環化反應。而Pisum sativum F125定點突變實驗中發現此點是影響酵素的結構以及受質的進出。

Oxidosqualene cyclases catalyze the oxidosqualene into tetracyclic or pentacyclic triterpeniods in the animal, fungi and high plants. A serial cyclization / rearrangement cascades catalyzed in one-step reaction by oxidosqualene cyclases have fascinated the bioorganic researcher over a half century. In 1992, Griffin proposed an aromatic hypothesis for stabilizing the respective carbocationic intermediates via “cation-π interaction” from the electron-rich indole ring of tryptophans, phenolic group of tyrosine, or phenylalanine residues within the cyclase active site. In order to clarify the mechanism of D ring expansion, the highly conserved aromatic amino acid residues in oxidosqualene cyclase, site-saturated mutagenesis experiments on phenylalanine125 residue of Pisum sativum(PSY) oxidosqualene cyclase which synthesis β-amyrin were carried out and then compare with the methionine105 residue of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase (ERG7). The protosta-20,24-dien-3β-ol products were isolated from the ERG7 M105G/A/S/N/Q/K mutants. This result indicated Met105 plays a catalytic role in the influence of rearrangement process and determination of deprotonation position but does not involve intervention in the cyclization steps. The function of Phe125 in Pisum sativum oxidosqualene cyclase is to stabilize the enzyme structure for movement of products.