- 學位論文
以化學救援法探討牛胰臟甲型去氧核醣核酸水解酶之活性區兩個組胺酸殘基在催化機制中所扮演的角色
Probing the catalytic mechanism of bovine pancreatic deoxyribonuclease by chemical rescue of inactive mutants of the essential histidine residues in the active site
摘要
牛胰臟甲型去氧核醣核酸水解酶（Bovine pancreatic DNase I，bpDNase）為研究最透徹的去氧核醣核酸水解酶，bpDNase為一醣蛋白，分子量約為31 kDa，其水解反應需要二價金屬離子如鈣、錳及鎂離子的催化，其中鈣離子的存在為酵素所必須，因為可穩定酵素結構及調節活性的功用。其最適作用條件為中性或弱鹼性環境，產物為帶有3’端羥基（3’-OH）、5’端磷酸根（5’-phosphate）之2~4個核苷酸聚合物。1992年Weston等人依據 bpDNase三級結構，提出可能的催化機制如下：His252扮演質子的接受者（general base），可以接受水分子的質子，之後水分子轉變成OH- 會攻擊DNA之磷酸雙酯鍵（phosphodiester bond）。另一方面，His134則扮演質子的提供者（general acid），將質子傳給DNA 3＇端O-，使磷酸雙酯鍵斷裂，而Asp212、Glu78可以穩定His252及His134。另Glu39可固定二價金屬離子，穩定整個水解過程。因此，依據三級結構推測His134與His252可能位在bpDNase之活性中心。 為研究這兩個活性區重要Histidine residues，除本實驗室現有之 brDNase (H134Q)與brDNase(H252Q)兩株突變型酵素外，另以PCR定位突變方法成功建構了四株突變型，分別為H134A與H252A，以Alaline取代Histidine；以及H134G與H252G，以Glysine取代Histidine，並以大腸桿菌BL21(DE3)pLysE表現重組蛋白。將破菌後的上清液以Source 15Q 陰離子交換樹脂進行管柱層析取得純化蛋白後，利用Metachromatic Agar Diffusion Assay觀察到兩者活性均相當低，僅為野生型bpDNase之10-4至10-5倍。我們嘗試以化學救援 (chemical rescue) 的方法對四株突變型酵素作進一步的研究，尋求以最直接的方式驗證前人所提催化機制之正確性，即添加各種不同濃度 (0~200 mM) 及不同pH值 (pH 5.5~8.0)的imidazole參與反應，試圖藉由外加的imidazole取代突變型H134A或H252A之histidine side-chain所喪失的imidazolium ring，執行催化酸鹼之反應，而能使突變型酵素之活性回復。實驗結果發現相對於野生型，0~100 mM imidazole在不同的pH值條件下，對於突變型H134A以及H252A之活性確實有不同程度的回復情形，分析結果並定出活性回復倍率。 為了進一步證實突變型的活性回復是因為外加的imidazole代替原本Histidine side-chain上的imidazole group執行了催化之功能，我們另外使用了氯化胺、甲基胺、乙基胺及丙基胺等四種pKa大小與分子大體積小不同的一級胺類，測試其對突變型酵素活性回復之影響。另外，我們也製備了突變株H134G及H252G，使其官能基移除後的空間更大，觀察此立體障礙之減少是否有助於外加的imidazole及其他的一級胺類對突變型酵素提供更顯著的活性回復作用。 本論文初步以化學救援法證實 H134與H252確實在bpDNase中分別扮演催化酸及催化鹼之角色。
關鍵字
化學救援法並列摘要
Bovine pancreatic DNase I (bpDNase), the most thoroughly studied endonuclease, is a glycoprotein of which the molecular weight is 31 kDa. Its hydrolysis requires divalent metal ions, such as calcium, manganese, and magnesium. The existence of calcium ion is essential for the enzyme because it can stabilize the structure of the enzyme and modulate the activity. The optimal condition for the activity is in a neutral or weak basic environment and the products are oligonucleotide with 5‘-phosphates (2~4 base). According to the 3D structure of bpDNase, Weston et al (1992) proposed the potential catalytic mechanism as follows. One histidine residue (H252) acts as a general base, abstracting a proton from the attacking water, whilst a second histidine residue (H134) functions as a general acid, protonating the leaving O3’ of substrate DNA. Consequently, based on the 3D structure, His134 and His252 are speculated to be located in the active site. In order to conduct a study on these two essential histidine residues in the active site, two existing mutants in our lab, brDNase (H134Q) and brDNase (H252Q), were investigated. Moreover, four other mutants were constructed using PCR site-directed mutagenesis: brDNase (H134A) and brDNase (H252A) in which alaline replaced histidine residues, as well as brDNase (H134G) and brDNase (H252G) in which glysine replaced histidine residues. By using “Metachromatic Agar Diffusion Assay”, it was observed that the activity of the H134A and H252A mutants is 10-5 to 10-4-fold lower as compared to the wild type. In an attempt to directly verify the validity of the catalytic mechanism proposed by the previous researchers, we performed further studies on the four mutants by chemical rescue. Imidazole of different concentrations (0~200 mM) and different pH values (pH 5.5~8.0) was therefore added to the reaction to replace the missing imidazolium ring in the histidine side-chain of the mutants H134A or H252A, to restore the activity of the mutants, and accordingly to execute the catalysis of general acid or general base. The results showed that, compared with the wild types, the addition of imidazole from 0 to 100 mM in concentration of different pH values did help restore the activity of the mutants H134A and H252A to various degrees. To further prove that the restoration of the activity is attributed to the addition of the imidazole that replaced the original imidazole group in the histidine side-chain in executing the catalysis, we also used four types of amine different in the pKa value and the molecular volume to test their effects on the restoration of activity to these mutants. In addition, the H134G and H252G mutants were constructed to enlarge their cavity in the active site, observing whether this abatement of the steric effect facilitates the restoration of activity to these histidine-mutants by the exogenously added imidazole and amines. By means of chemical rescue, this study confirmed that H134 and H252 indeed play the role of catalytic general acid and general base, respectively, in the catalysis of bpDNase.