醯亞胺水解酵素的反應機制

Abstract

醯亞胺水解酵素是一個受質廣泛的解毒酵素，催化大部分含有醯亞胺官能基的化合物，然而其特異性與機制一直不很了解，尤其不同來源的醯亞胺水解酵素更有著顯著的差異。本論文內容便是以細菌、魚類與哺乳動物來源的醯亞胺水解酵素做一有系統性的探討，並主要針對他們的基本性質、受質選擇性與特異性、嗜熱性與嗜冷性質、輔因子金屬的特異性、化學反應機制差異、轉譯後修飾與分子結構、新官能基受質的發現等等，並分為八個章節敘述之。首先，我們建立了不同來源醯亞胺水解酵素的表達與純化方法。第二，溫度的調控對穩定性與受質選擇性有著顯著的差異。第三，輔因子金屬的不同使得活性、穩定性與受質特異性也產生相當的影響。第四，不含醯亞胺官能基的新受質的發現。第五，轉譯後修飾的機制與輔因子金屬的關聯性。第六，不同來源醯亞胺水解酵素的結晶與分子結構的獲得。綜觀以上的研究，這些結果構成了第一個發現新型嗜冷性的醯亞胺水解酵素、新受質的發現、轉譯後修飾與金屬分子的化學反應機制，並因而開啟了許多待探索的領域。

Imidase is an enzyme catalyzing many imides. Previously, specificity and mechanism of imidases remains poorly understood, especially some significant variations are found in substrate specificity, metal content, amino-acid sequence, subunit composition but thermal properties from different sources. In this project, these basic questions such as substrate selectivities and specificities, thermophilicities and psychophilicities, specificities and contents of the cofactor metal(s), chemical reaction mechanisms, posttranslational modification and molecular structure, and novel substrates bearing new functional group of varying imidases were studied among fish, bacterial and mammalian, and described into eight chapters. First, we established the convenient expression or purification procedures of fish, bacterial and mammalian imidase. Furthermore, we found the following observations: (1) temperature could regulate substrate selectivity of imidases; (2) metals might regulate the activity, stability, and specificity of imidases; (3) new substrate of imidases without imide group; (4) the function-structure relationship between the posttranslational modification of carboxylated lysine and metals of bacterial imidase; (5) crystals of bacterial and mammalian imidases. These results constituted a first systematic study for exploring of a novel cold-adapted imidase, new substrates, mechanisms of metals action and posttranslational modification, and therefore initiated many interesting fields.