人類岩藻醣苷水解酶之抑制研究

Abstract

人類岩藻醣苷水解酶（Human α-L-fucosidase，AFU）屬於醣類水解酵素第29家族的水解酶（EC 3.2.1.51），本研究著重於該酵素的相關催化功能與抑制作用的探討。 將建構在pET22b上的Human α-L-fucosidase基因轉殖進入大腸桿菌BL21（DE3）中表現；經過離子交換管柱（SP、Q）及G-75凝膠管柱的純化處理之後，可以取得均質度達95％的酵素，酵素單體的分子量約為50 kDa左右。以p-nitrophenyl-α-L-fucopyranoside (PNPF) 為基質進行活性分析，實驗顯示AFU在pH 4.5以及pH 6.5左右存在兩活性最佳區域；當pH低於3.5或是高於7.5後酵素會呈現不穩定狀態。以PNPF為反應基質在pH 5.0的環境下，其Km以及kcat分別為0.105 mM和48.6 s-1；相較文獻中人類肝臟萃取的AFU （Km = 0.43 mM，Vmax = 19.6 μmole/mg/min 相當於kcat = 16.3 sec-1），重組酵素的催化能力（kcat/Km）約為肝臟原生酵素的12倍。 為研究酵素反應之機制，我們利用化學合成法合成一系列具有不同離去基之基質進行Brǿnsted relationship之研究，以離去基pKa 大於7.0為基質，得到Brǿnsted constant（βlg）約為-0.27；又因酵素進行催化反應時也觀測到initial burst 現象，推測其催化作用應涉及兩步驟雙取代之機制，且速率決定步驟應為去醣基（deglycosylation）步驟。 本實驗亦進行藥物篩選之研究，利用ELISA reader從藥物庫[Spectrum Collection（MicroSource）]中進行藥物篩選；我們幸運的篩選得到幾種有效之AFU抑制劑分別為，不可逆抑制劑：Cisplatin、Ebselen；競爭型抑制劑：Ethambutol、Mitoxantrone及非競爭型抑制劑：Dequilinium chloride。Cisplatin以及Ebselen推測會與蛋白質中的半胱胺酸（cysteine）形成共價鍵結，使蛋白質結構產生變化或破壞催化區而失去催化活性。競爭型抑制劑Ethambutol對AFU的抑制常數（Ki）為23 μM；以Ethambutol對不同的醣類水解酵素及不同來源之AFU進行抑制研究，發現Ethambutol對人類AFU具有較佳的專一性；此抑制劑為目前治療肺結核第一線藥物之一。

A gene from human encoding α-L-fucosidase (AFU) was cloned into pET22b plasmid. Protein was successfully expressed in E. coli BL21 (DE3). After applying a series of ion-exchange and gel-filtration chromatography purification steps, recombinant AFU with 95% homogeneity can be obtained. The molecular weight of the enzyme was analyzed by SDS-PAGE to be about 50 kDa. pH-dependent study indicated that AFU exhibited 2 optimal regions at pH 4.5 and pH 6.5, and the enzyme would become unstable when pH is lower than 3.5 or higher than 7.5. Michaelis constant (Km) and catalytic activity were determined with p-nitrophenyl-α-L-fucopyranoside (PNPF) and were found to be 0.105 mM and 48.6 sec-1, respective. Comparing with AFU extracted from human liver (Km = 0.43 mM and maximal velocity = 19.6 μmole/mg/min equal to kcat = 16.3 sec-1)，the catalytic power (kcat/Km) of recombinant AFU is 12-fold stronger than native human liver AFU. In order to investigate the reaction mechanism of AFU, a series of aryl-α-L-fucopyranoside were synthesized for Brǿnsted relationship study. The Brǿnsted constant βlg is -0.27 obtained from Brǿnsted plot constructed with a series of aryl-α-L-fucopyranoside with pKa > 7.0. Initial burst also observed during the enzyme reaction. Based on these two preliminary results, the catalytic mechanism of AFU was purposed to be a two-step double displacement mechanism with the rate-limiting step at the deglucosylation step. Inhibitor of AFU was also screened. A chemical library from Spectrum Collection® （MicroSource）was used as drug candidate for screening. Fortunately, several inhibitors were found to be effective such as irreversible inhibitor: Cisplatin, Ebselen; competitive inhibitors: Ethambutol、Mitoxantrone and uncompetitive inhibitor: Dequalinium chloride. Cisplatin and Ebselen would covalently bond to the amino acid residue cysteine of AFU; hence, the active site structure of the enzyme may be changed and finally lost its activity through this bonding. The Ki value of Ethambutol for human AFU was 23 μM； specific inhibition study for different glycoside hydrolases and AFU from different sources with Ethambutol was also investigated. Ethambutol is also one of the first-line medications for pulmonary tuberculosis, was found to be a specific inhibitor of recombinant human AFU.