Abstract
E.coli中的methionine aminopeptidase(MAP)為一種需要兩價鈷離子活化的金屬酵素。它主要的功用，在切除細胞內轉譯過程後新合成蛋白序列中的胺基端的第一個胺基酸methionine。由已經發表的E.coli MAP X光繞射結構分析得知，在MAP中的Asp 97，Asp 108，His 171，Glu 204， Glu235 五個胺基酸以離子鍵結的方式，形成近似八面體結構的鍵結；而在鈷離子鍵結區域附近，存在一個由Cys 59，Cys 70，Tyr 62，Tyr 65，Phe 177， Trp 221 六個胺基酸所構成的疏水性袋區，這個區域被推測是 MAP進行酵素催化反應時受質鍵結最有可能的地方。 本次研究中我們利用定點突變的技術分別改變 MAP上五個和鈷離子鍵結的胺基酸特性，在寄主細胞中大量表現後，純化出這些突變的MAP。測量這些突變MAP的酵素活性發現，這些和鈷離子鍵結相關的胺基酸的突變 MAP，其酵素活性已經喪失。進一步檢測突變MAP中的鈷離子含量，也發現在這些突變MAP中已經無法測量到鈷離子的存在。 據此推論，MAP中這五個和鈷離子鍵結相關的胺基在維繫兩個鈷離子的穩定鍵結，及 MAP的酵素活性上，都有著無可或缺的重要性。 除此之外，本次研究中也針對疏水性區域Cys 70及Trp221兩個突變MAP進行酵素動力學的評估。結果顯示，Cys 70及Trp 221突變之後會降低酵素催化過程中的Vmax，並使得Km值上升，顯示這兩個位置的突變，已經對催化過程中酵素與受質的親合力造成影響。因此我們推測，疏水性袋區在 MAP的催化過程中確實扮演著鍵結受質的重要角色，而在構成疏水的六個胺基酸中，又以Cys 70及Trp 221最為重要。Escherichia coli methionine amino peptidase (MAP) is a cobalt-dependent metallo-enzyme which removes amino-terminalmethionine from nascently synthesized peptides. Crystalstructure of MAP protein has shown that there are two cobaltions ligandig with the side chains of Asp 97, Asp 108, His 171,Glu 204, and Glu 235. In addition, it reveals a hydrophobicpocket which may be the substrate binding site of the enzyme.In this study, site-directed mutagensis was conducted toinvestigate the significance of these amino acid at thehydropobic pocket and the cobalt binding sites. The mutatedgene products were expressed in E.coli and the recombinant MAPwas purified to near homogeneity by DEAE-Sepharose anionexchange, Superose 12 gel filtration and Mono-Q anion exchangecolumn chromatography. The results demonstrated that MAPactivity drops to ground level when mutation occurred at any ofthe cobalt binding site as compared with that of the wild type.Examination of the cobalt content in these mutated MAPsrevealed that there was nearly no cobalt ions inside thesemutated proteins. MAP activity of the C70S and W221L mutants atthe hydrophobic pocket droped to less 50% of the wild type.Mutations at both positions almost reduced the activity toground level. Comparing the kinetic parameters of C70S and W221with that of wild type, an increase of Km and a decrease ofVmax were noted. These results indicate that all of the aminoacid residues at the metal binding site are important inmaintaining the integrity of the MAP activity and the stabilityof the two cobalt ions inside the MAP. As for the mutations atthe hydrophobic pocket, altering Cys 70 and Trp 221 these twoamino acid residues might cause the weaker binding betweenenzyme and substrate. It is suggested that Cys 70 and Trp 221play an influential role for the enzyme-substrate recognitionand binding.