牛肝中氧化鯊烯-羊毛硬脂醇環化酵素之生化特性與抑制作用的探討

Abstract

(氧化)鯊烯環化酵素 (EC 5.4.99.-) 是由一群能夠催化具有多樣性和複雜性的環化/重組反應的酵素所組成，可將受質鯊烯或是氧化鯊烯經由循序性過程，轉變為一系列多環結構的固醇類及三萜類產物。牛肝中氧化鯊烯-羊毛硬脂醇環化酵素 (OSC) 可以由細胞微粒體中收集得到，並藉由超高速離心、Q-Sepharose離子交換、Hydroxyapatite及HiTrap Heparin等三種管柱層析方法，純化酵素至均質態。在酵素纯化過程中需要加入介面活性劑Triton X-100來維持其最佳的活性；利用SDS-PAGE電泳分析後，以commassie blue染色，可得到分子量約為70 kDa的單一蛋白質帶。牛肝中氧化鯊烯環化酵素的生化特性，顯示其催化反應的最理想酸鹼值與溫度條件分別為pH 7.4與37 °C。酵素 動力學參數KM 與 kcat / KM 經過估算後分別為11 μM 與1.45 mM-1min-1；而抑制物Ro48-8071對於環化酵素具有顯著的抑制效果，且IC50值可達到僅11 nM。由DTNB修飾反應及DTNB與Ro48-8071對於酵素共同競爭作用的結果綜合而言，推論可能有2個半胱胺酸與酵素的催化功能有關，然而其位置並非位於受質結合或酵素催化活性區域內。DEPC對於環化酵素所產生的去活化作用，以在接近中性的環境下最為明顯；此外，由於HA能再度回復酵素活性，也可證明化學修飾反應的位置確實是在組胺酸上。而從計量化學及DEPC產生的去活化作用判斷，每一單位的氧化鯊烯環化酵素活性區域內，可能有2個組胺酸位置對於酵素的催化功能是重要的。

(Oxido)squalene cyclases (EC 5.4.99.-) constitute a family of enzymes that catalyze the diverse and complex cyclization/rearrangement reactions of a squalene or oxidosqualene to a distinct array of polycyclic sterols and triterpenoids. The oxidosqualene-lanosterol cyclase (OSC) from bovine liver has been isolated from the microsomal membrane and purified to homogeneity by ultracentrifugation, Q-sepharose, Hydroxyapatite and HiTrap Heparin chromatographies. The purified enzyme required detergent Triton X-100 to retain its highest activity. The cyclase had a molecular mass of ~70 kDa, as evidenced by a single protein band on coomassie-stained SDS-PAGE. The purified cyclase showed pH and temperature optima at pH 7.4 and 37 °C, respectively. The apparent kinetic KM and kcat / KM values were estimated to be 11 μM and 1.45 mM-1min-1, respectively. Inhibition study using Ro48-8071 showed a potent inhibition of OSC with an IC50 of 11 nM. Results from DTNB modification and DTNB coupled with Ro48-8071 competition study suggest that two sulfhydryl groups are involved in the catalysis but not located in the substrate binding pocket or catalytic active site. The purified OSC was maximally inactivated by DEPC near neutral pH and re-activated by hydroxylamine, indicating the modification of histidine residues. The stoichiometry of histidine modification and the extent of inactivation showed that two essential histidine residues per active site are necessary for bovine liver OSC activity.