蝦肝胰臟核酸水解酶活性中心之重要胺基酸殘基對酵素活性影響之研究

Abstract

蝦肝胰臟核酸水解酶（shrimp hepatopancreatic nuclease, 簡稱 shNuclease），以前被認為是一種 DNase Ι，後來發現它在鎂離子及鈣離子的存在下，具有水解 RNA 的活性，因此更名為 nuclease。ShNuclease 是由21個胺基酸的信號胜肽及 381 個胺基酸的成熟蛋白質組成。它含有十一個半胱胺酸，其中十個形成五對分子雙硫鍵。 ShNuclease 並沒有同質性蛋白質，但它的第 205 到 255 個胺基酸序列和 Serratia marcescens 等數種生物的核酸水解酶具有保留性。之前已有報導證實，Serratia marcescens的 nuclease（簡稱Serratia nuclease）之三級結構中的 Arg57、Arg87、His89、Asn119 及 Glu127 等胺基酸殘基在催化上扮演重要角色；對應到 shNuclease，分別為 Asn179、Lys209、His211、Asn241 及 Glu249，且His211為 shNuclease 的活性中心。 本實驗室先前已成功建立 shNuclease 在人類293T 細胞的表現系統，並構築 K209R、K209L、H211Q、E249D 及E249Q 等五株突變株，發現除了 K209R 的活性增加為野生型的 1.7 倍，其餘突變株的活性皆大幅下降[33]。推論 His211 及 Glu249 可能直接參與催化反應，而 Lys209 可能和對應於 Serratia marcescens 的 Arg87 一樣，為受質結合殘基。所以當突變為較鹼性的 Arg 後，變得較易和受質結合而使酵素活性增加。 為了進一步研究 shNuclease 的催化機制，我們另外建立了 N179A、N179R、N179D、K209A、N241D 及 N241Q 等六株突變株。同樣地利用人類 293T 細胞表現後收取細胞培養液，經由濃縮後，再利用 Ni-NTA 膠體進行純化。將純化後的野生型及各突變株shNuclease 進行西方墨點分析及活性染色，發現 N179A 的比活性只有野生型的 70 ％，N179R 的比活性則上升為野生型的 158 ％，而 N179D 比活性降為野生型的 59 ％。相對於 shNuclease Asn179 為 Serratia nuclease Arg57，又由之前 Friedhoff 等人證實， Arg57 在 Serratia nuclease 中扮演穩定過渡狀態的角色，故推測 Asn179 在 shNuclease 中可能也扮演相同的角色。因此將 Asn179 突變為 Arg 後，所帶的正電較有利於過渡狀態的穩定，造成酵素活性上升。若突變為帶負電的 Asp，較突變為同樣不帶電的 Ala 會使酵素活性下降更多。而 K209A 偵測不到活性，顯示 Lys209 可能藉由其所帶的正電與 DNA 結合，並對催化反應具有一定的重要性。另外 N241D 及N241Q 也都不具活性，顯示出 Asn241 的不可替代性。推測 Asn241 可能和對應於 Serratia nuclease 的Asn119一樣，具有穩定過渡狀態的功能，或為必要輔因子 Mg2+ 的 ligand。由於我們的實驗結果和 Friedhoff 等人相當接近，推測 shNuclease 的催化機制可能和 Serratia nuclease 相當類似。

Shrimp hepatopancreatic nuclease（shNuclease）, previously designated as DNase I, has RNase activity in the presence of Mg2+ and Ca2+ ions. The shNuclease precusor contains 21-residue signal peptide and a 381-residue mature protein. The enzyme has 11 cys residues, forming five intramolecular disulfide bonds. Sequence alignment revealed there is no homologous protein. However, residues 205-255 shared a conserved active-site motif within a distinct group of nucleases, including Serratia marcescens. According to the previous report, Arg57、Arg87、His89、Asn119 and Glu127 of the nuclease of Serratia marcescens（Serratia nuclease）are very important in catalysis. And the five critical residues of Serratia nuclease are equivalent to Asn179、Lys209、His211、Asn241 and Glu249 of shNuclease. His211 showed to be in the centre of active site. In our previous studies, the recombinant shNuclease expression system was established and the K209R、K209L、H211Q、E249D and E249Q variants were constructed. The expressed proteins showed that the specific activities of K209R increased 1.7-fold as that of wild type shNuclease, while those of other variants decreased drastically, indicating His211 and Glu249 could be directly involved in catalysis. Lys209 could be the substrate binding residue in shNuclease corresponding to Arg87 in Serratia nuclease. Because Arg has stronger basicity than Lys, replacing Lys209 with Arg may promote the substrate binding ability of shNuclease. Therefore the DNase activity of K209R in shNuclease was increased [33]. To further investigate the catalytic mechanism, we constructed N179A, N179R, N179D, K209A, N241D and N241Q variants. Wild type and mutated recombinant shNucleases were expressed in human 293T cell. The recombinant shNuclease in the culture media was collected and purified with Ni-NTA agarose beads. Based on the intensities on Western blots and zymograms, the specific activity of N179A, N179R and N179D variants were 0.7-fold, 1.58-fold and 0.59-fold as compared with that of wild type shNuclease, respectively. Friedhoff et. al proposed that Arg57 of Serratia nuclease participated in transition state stabilization. Since Arg57 of Serratia nuclease is equivalent to Asn179 of shNuclease, we suggested that Asn179 might play the same role. When replacing Asn179 with Arg, the positive charge of Arg may promote the transition state stabilization, therefore the DNase activity of N179R shNuclease was increase. The negative charge of Asp may reduce the DNase activity of shNuclease more than the uncharged Ala residue. Because K209A had no DNase activity, we suggest that Lys209, which is positivily charged, may bind DNA in shNuclease. Because N241D and N241Q had no DNase activities, we suggested that Asn241 might participate in catalysis. Like Asn119 in Serratia nuclease, Asn241 might participate in the transition state stabilization or involed in ligand binding of the essential cofactor, Mg2+. Our results were similar to that of Friedhoff et. al. Therefore we proposed that the catalytic mechanism of shNuclease might be similar to Serratia nuclease.