醯亞胺水解酵素催化反應機制-黑青斑河豚之醯亞胺水解酵素分子選殖、表現以及其功能性研究

Abstract

脊椎動物之醯亞胺水解酵素是一個具有單一金屬中心的酵素，其在生理上的功能是參與代謝uracil和thymine過程中的第二個酵素。此酵素除能夠水解生理上的受質-dihydropyrimidines外,亦能催化其他許多含有醯亞胺官能基的化合物。在本研究中，由黑青斑河豚中選殖出來的醯亞胺水解酵素基因可利用大腸桿菌表現系統進行酵素表達。我們從添加1 mM 鈷離子於培養基中並純化出此酵素，經由ICP-MS分析，每一個酵素的單體約含有0.83，0.08，和0.07個鈷，鐵，及鋅離子；而在不添加金屬離子的培養基中所純化出的酵素中則發現含有大量的鐵離子 (每個單體含0.77個鐵離子)。從添加鈷離子培養基中純化出的酵素之比活性較從不添加金屬離子的培養基中純化出的酵素活性高約23倍之多，因此我們推測含有鐵離子的醯亞胺水解酵素具有極低的活性或是根本不具有活性。由胺基酸序列分析顯示黑青斑河豚的醯亞胺水解酵素的序列分別和人類以及大鼠的序列有73 % 以及 72 % 的相似度。另外，由多重序列比對可發現：在具有雙金屬中心的hydantoinase中，與金屬離子形成配位的六個胺基酸也同時保留在脊椎動物的醯亞胺水解酵素序列中。在酵素特性上，黑青斑河豚的酵素單體分子量約為54 KDa，且為一個四聚體；其對生理上的兩個受質的酵素動力學常數Km遠小於其他異生受質(xenobiotic substrate)。而由熱穩定的實驗中顯示黑青斑河豚的酵素較豬肝之酵素不穩定，其在50℃下反應2小時後，喪失了60% 的酵素活性。綜合以上的結果，此為第一個有關脊椎動物之醯亞胺水解酵素在原核生物系統表達之報導，由此可提供我們一個研究平台去探究具有一個金屬中心之醯亞胺水解酵素的催化機制。

Imidase from vertebrates is a mononuclear metalloenzyme that catalyzes the reversible hydrolysis of dihydropyrimidines and many other imides. Recombinant T. nigroviridis imidase purified from E. coli cell cultures amended with 1 mM cobalt is found to process 0.83 cobalt, 0.08 Fe and 0.07 Zn per subunit, whereas iron (0.77 Fe per subunit) is the major amount of metal in enzyme purified from non-metal amended cell cultures. The specific activity of imidase isolated from cultures grown in the presence of 1 mM cobalt is about 23-fold higher than that without additional metal ions added. We propose that iron-containg form of imidase is partially or fully inactivated. Analysis of the amino acid sequences reveals that this novel T. nigroviridis imidase displays 73% and 72% sequence identities with those of human and rat, respectively. Multiple sequence alignments show that the ligands to the metal centers in hydantoinase appear to be conserved in all known vertebrate imidase, even including the bridging carboxylated lysine residue. The isolated T. nigroviridis imidase is a homotetrameric enzyme and the subunit molecular mass is 54 KDa. The enzyme purified from cobalt supplemented cultures processes very low Km values for two physiological substrates, dihydrouracil and dihydrothymine, as compared to those of xenobiotic substrates. A thermostability experiment reveals that T. nigroviridis imidase is less stable than the thermophilic pig liver imidase and loses 60% of its activity in 2 hr at 50℃. These results constitute the first report about the vertebrate imidase expressed in a prokaryotic system and provide us the tool to study the detailed catalytic mechanism of vertebrate imidase with a mononuclear metal center.