豬肝醯亞胺水解酵素在不同pH和溫度對基質特異性的影響

Abstract

嗜熱性豬肝醯亞胺水解酵素(imidase)是一廣效性的解毒酵素，由於它能催化水解多種的基質，亦被稱為dihydropyrimidinase (EC.3.5.2.2)和hydantoinase。我們從豬肝中萃取出純的酵素，以便進行之後的實驗。我們的研究目的是想要瞭解在不同pH和不同溫度條件環境下，醯亞胺水解酵素對基質的選擇性的影響。我們發現在室溫下對hydantoins類的衍生物而言，酵素活性會隨著衍生物所帶的取代基立體障礙增大而減小，但是在高溫則不一定。而帶有5,5-雙取代基或NH2-取代基都觀察不到活性。我們將基質大致分為三大類：五環的hydantoins類，六環的dihydropyrimidines類和五環及六環的cyclic imides類，其最適的pH值分別為8.0，9.5，8.5。六環的cyclic imide和dihydropyrimidines具有相似形狀Km的pH趨勢圖，而不同於五環的cyclic imide或hydantoin，故Km的pH趨勢圖是與基質的環狀結構相關的。而cyclic imides和hydantoins類Vmax的趨勢圖是呈現出鐘形曲線，與dihydropyrimidines類不一樣。酵素的特異性也是與溫度相關的，在不同溫度條件下，cyclic imides和hydantoins類的pH趨勢圖都相似，而dihydropyrimidines類則否。隨著溫度的不斷上升，六環的dihydropyrimidines和glutarimide的Km與Vmax值也是隨之增加，但是五環的hydantoin和succinimide的Km與Vmax值則不受溫度變化的影響。故我們可結論出在不同pH與溫度條件下，基質取代基大小與基質環狀結構(五環或六環)和酵素的基質特異性具有相當大的影響。

Due to its broad substrate specificity, thermophilic imidase is also known as dihydropyrimidinase (EC 3.5.2.2) and hydantoinase. The purpose of this research is to understand the effect of temperature and pH on the substrate specificity of imidase. At room temperature, hydrolytic activity of imidase decrease toward hydantoins as its size of side chain increase. Opposite results are observed at 60 ℃, in which condition hydantoins with large side chain become much better substrates. Three functional groups of substrate include six and five-member ring are used for this study. The optimal pHs of imidase are 8.0, 8.5, 9.5 for Vmax/Km of hydantoins, cyclic imides and dihydropyrimidines, respectively. Effect of pH on Vmax and Km is dependent on the substrate used. Km of a six-member ring cyclic imide and dihydropyrimidines are insensitive to the change of pHs from 7.5 to 9.5. The pH profile of Vmax of cyclic imides and hydantoins exhibit bell shapes profile, which is different from that of dihydropyrimidines. Substrate specificity of imidase for imides, hydantoins and dihydropyrimidines is temperature dependent. The shapes of pH profiles may remain similar (hydantoins and cyclic imides) or may differ (dihydropyrimidines) at different temperatures. Both Km and Vmax increase steadily with the elevation of reaction temperature up to 60 ℃ for dihydropyrimidines and glutarimide but not for hydantoins and succinimide. We conclude that ring structure and side chain are both important for the substrate selection of imidase at different temperature and pH.