牛胰臟去氧核醣核酸水解酶中短距雙硫鍵之似硫氧化還原素活性與鈣離子結合能力之研究

Abstract

牛胰臟去氧核醣核酸水解酶 (bpDNase) 為目前被研究的最透徹之一種DNase。BpDNase由260個胺基酸所組成，分子量為31 kDa，在其分子中具有2對雙硫鍵，C173-C209與C101-C104。由之前的研究中，已知C173-C209這對長距雙硫鍵與維持整個酵素構形有關，如被還原會使酵素失去活性；反之若是短距雙硫鍵C101-C104被還原，則不會影響酵素活性，因此長久以來bpDNase中這對短距雙硫鍵C101-C104即被定義為非必需雙硫鍵。然而，經由DNase I序列的比對，可以發現這對短距雙硫鍵仍相當保守地存在於各物種間。同時，由以結構為基礎之序列比對，亦發現這對短距雙硫鍵與硫氧化還原素同樣具有一段CXXC motif，兩者在立體空間結構上也有類似的地方。因此，對於這對短距雙硫鍵在bpDNase中究竟扮演何種角色，我們感到相當有興趣。我們希望了解是否C101-C104這對非必需雙硫鍵存在與否雖不會影響酵素活性，卻可以在bpDNase中扮演水解DNA之外的其他角色，因而可以相當保守地存在於各不同物種之DNase中。 經本實驗室陳威戎博士以實驗初步證實還原態之bpDNase的確具有似硫氧化還原素之活性後，我們繼續利用定位突變的方法，分別建構了四株雙重突變株 (E102G/S103P、E102P/S103G、G100K/G105W、G100W/G105K)，與二株四重突變株 (G100K/E102P/S103G/G105W、G100W/E102G/S103P/G105K)。我們首先將這幾株突變酵素以E.coli strain BL21(DE3)pLysE大量表現後，分別通過陰離子交換管柱 SOURCE 15Q及陽離子交換管柱 S HyperD，並以SDS-PAGE分析和硝酸銀染色結果確定得到純化至均質的產物。藉由蛋白質定量與DNase活性分析之結果計算各株重組牛胰臟去氧核醣核酸水解酶突變酵素之比活性，發現除四重突變株 (G100K/E102P/S103G/G105W) 之比活性為野生型酵素之一半外，其餘幾株重組蛋白之比活性皆沒有顯著改變。配合圓二色偏光光譜進行重組蛋白之二級結構分析，亦證實除四重突變株 (G100K/E102P/S103G/G105W) 之結構中β-摺板含量偏高，與原態bpDNase差異較大外，其餘各突變株之結構皆沒有產生太大改變。 接著我們以胰島素還原沉澱法觀察這些重組蛋白之似硫氧化還原素活性，結果顯示當我們將bpDNase之短距雙硫鍵附近部分區域突變為與硫氧化還原素活性區類似之序列後，確實可以提升重組蛋白之似硫氧化還原素催化活性。尤其是將序列完整突變為與硫氧化還原素活性區完全相同之正向四重突變株 (G100W/E102G/S103P/G105K)，其似硫氧化還原素活性可由原本野生型酵素之11.3 % 大幅提升至66 %。 另外，由於這兩對雙硫鍵所在區域恰對應於bpDNase之鈣離子結合位置，site I和site II，而結合在site II之鈣離子又恰與負責固定短距雙硫鍵Cys101-Cys104所在之鬆散loop有關。我們進一步以實驗觀察，發現在我們改變了bpDNase中短距雙硫鍵附近的區域後，唯有反向四重突變株 (G100K/E102P/S103G/G105W) 對鈣離子之結合能力特別弱，推測可能與其二級結構上之改變有關。

Bovine pancreatic deoxyribonuclease (bpDNase) is the best-characterized DNase. It is composed of 260 amino acids, and is a glycoprotein with a molecular weight of 31 kDa. One large (C173-C209) and one small (C101-C104) disulfide loops were found in bpDNase. Earlier studies showed that the large disulfide loop was responsible for the enzyme conformation. When the large disulfide loop was reduced, bpDNase lost its enzyme activity. In contrast, reduction of the small loop resulted in an enzyme with the full activity. However, sequence alignment of DNases from various species revealed that this small loop was still highly conserved among species. Moreover, because the structure-based sequence alignment revealed homology between the small disulfide loop and the active site motif (-CXXC-) of thioredoxin, we are interested in seeking the possible biological roles of the small loop in bpDNase. Although not related to enzyme activity, the nonessential disulfide C101-C104 might be very important for other unknown functions. The importance of this disulfide also can be found in conservative sequences of various DNases. According to our recent studies, the reduced bpDNase actually contained the thioredoxin-like activity based on the rate of insulin precipitation assay. In order to gain further insight into the biological functions of the small loop, four double (E102G/S103P、E102P/S103G、G100K/G105W、G100W/G105K) and two quadruple (G100K/E102P/S103G/G105W、G100W/E102G/S103P/G105K) mutants were constructed using site-directed mutagenesis. Recombinant proteins were expressed in E. coli strain BL21(DE3)pLysE and were purified through a SOURCE 15Q anion and a S HyperD cation-exchange columns. SDS-PAGE with silver stain confirmed the homogeneity of the purified brDNase variants. Most of the recombinant proteins possess similar specific activities as the wild type bpDNase. However, quadruple mutant KPGW exhibited only half of the activity. CD spectra analysis also revealed significant different for this mutant. In our studies, we found that all these brDNase variants were able to accelerate the rate of insulin precipitation. And the highest thioredoxin-like activity (66%) of the quadruple mutant WGPK suggests that the conserved sequence (-WCGPCK-) of thioredoxin is crucial for its activity. Previous studies also showed that these two disulfide bonds were correlated with the two calcium binding sites of bpDNase, site I and site II. It was shown that the binding of calcium of site II is responsible for the conformation of the loose loop, C101-C104. We found that among brDNase variants which were mutated around the small loop only the reversed-sequence quadruple mutant KPGW presented a weaker binding ability, probably due to the alteration of its secondary structure.