Abstract
甲硫胺酸胺基水解酵素 (簡稱 MAP ) 是一種需要鈷離子的參與,才具有活 性的金屬酵素. 其在細胞內的生理角色，是能專一性地移走大部份初生成 蛋白質的起始甲硫胺酸, 使這些蛋白質能夠進一步被修飾, 而有正常的功 能. 雖然在真核生物及原核生物中, 都有 MAP 的發現, 但是 MAP 的功能 和其構造上的關係, 到目前為止還不是很清楚. 從大腸桿菌分離出的 MAP,經 X - 光繞射結構分析得知,有一個疏水性區域是由兩個半胱胺酸 ( Cys59,Cys70 ),兩個酪胺酸 ( Tyr62,Tyr65 ),一個苯丙胺酸 ( Phe177 ),及一個色胺酸 ( Trp221 ) 的側鏈所組成,可能是 MAP 和其受 質結合並進行催化作用的所在.我們首先以定點突變法改變這兩個位置的 半胱胺酸,在讓突變的蛋白質表現後，我們純化此蛋白質並進行其酵素活 性的測定.接著我們亦逐一突變在這個疏水性區域中其餘的胺基酸. 比較 正常和經突變的酵素活性得知，Cys70 及 Trp221 這兩個胺基酸的變異， 會導致 MAP酵素活性的降低。因此我們認為這個疏水性區域內的上述二胺 基酸，參與了大腸桿菌 MAP 的活性。另外，我們也發現大腸桿菌 MAP 的 羧基端胺基酸在 MAP 的催化作用上亦可能佔有重要的地位. Methionine aminopeptidase (MAP) is a cobalt-dependent metalloenzyme which specifically cleaves the initial methionine from nascently synthesized proteins. MAP has been studied in pro karyotes and eukaryotes, but the structural and functional rela- tionship of the enzyme is not clear. For Escherichia coli MAP, a hydrophobic pocket lined by side chains of Cys59, Cys70, Tyr62, Tyr65, Phe177 and Trp221 on MAP was predicted from the X- ray cry stal structure as the region for substrate recognition and cata- lysis. We attempted to determine the active site of the enzyme by changing each amino acid in the predicted hydrophobic pocket by site - directed mutagenesis. The mutated proteins were expre- ssed and purifed and their activities were compared with that of wild type MAP. The results showed that mutations at Cys70 and Trp221 dramatically decreased MAP activity. It is concluded that Cys70 and Trp221 in the predicted hydrophobic pocket may be the amino acids involved in the enzyme activity. Additionally, we also discovered that the carboxy-termunal amino acids of E. coli MAP may play an important role for the activity of E. coli MAP.