臺灣博碩士論文加值系統

3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) from Comamonas testosteroni, a member of the short-chain dehydrogenase/reductase (SDR) family, catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH. Previously studies have showed the residue Tyr155 acts as a general base in the 3α-HSD/CR catalyzed reaction. Structurally, 3α-HSD/CR is a homodimer. A salt-bridge interaction is formed between the Asp249 in helix αCT of each subunit with Arg167 in helix αF of other subunit based on structural analysis. To explore the structural and functional role of Arg167 in the oligomerization of 3α-HSD/CR, we performed site-specific mutagenesis to replace Arg167 to Ala, Gln and Asp, respectively, and also utilized kinetic assay, spectroscopic measurment, gel filtration chromatography, analytical ultracentrifugation, thermal and urea unfolding experiments. In kinetic assay, kcat/Km of R167A, R167Q and R167D were drastically decreased by 1.5×105-fold,1.5×104-fold and 6×105-fold compared with wild-type, respectively. The secondary structures of the wide-type and mutants of R167A, R167Q, and R167D are slightly different based on the results from the CD spectra, while a red shift is observed in the mutants on fluorescence spectra.The results from gel filtration chromatography and sedimentation velocity indicate a concentration-dependent dimerization of wide-type and mutant enzymes. The study from the thermal and urea unfolding indicates the mutation of the residue Arg167 destabilized the protein structure. In conclusion, the salt-bridge interaction between Asp249 and Arg167 of each subunit is important in stabilizing dimeric formation of 3α-HSD/CR.

表目錄……………………………………………………B
圖目錄……………………………………………………C
中文摘要…………………………………………………E
英文摘要…………………………………………………G
壹、前言…………………………………………………1
貳、實驗分法與材料
一、藥品與試劑…………………………………………7
二、緩衝溶液製備………………………………………9
三、主要儀器與器材……………………………………11
四、實驗方法
1.突變型3α-HSD/CR質體(pET15b-3α-HSD/CR)製備…12
2.重組野生型及突變型3α-HSD/CR之純化……………15
3.酵素動力學參數測定…………………………………18
4.圓偏振二旋圖譜分析…………………………………18
5.螢光圖譜分析…………………………………………18
6.膠體過濾層析…………………………………………19
7.超高速離心沉降速率分析……………………………20
8.蛋白質結構熱變性實驗………………………………20
9.尿素影響蛋白質結構實驗……………………………21
参、實驗結果……………………………………………23
肆、討論…………………………………………………30
伍、參考文獻……………………………………………34

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