探討蛋白質四級結構對於細胞質亞硫酸基轉移酵素之催化活性與功能的影響

Abstract

亞硫酸基轉移酵素目前大部分已知為同型二聚體，在生理上負責將生物分子亞硫酸化，藉此調控許多重要的生理反應。本論文主要在探討四級結構對此酵素活性的影響，因此，藉由膠體過濾分析實驗的證實，我們成功地製備人類酚亞硫酸基轉移酵素及脫氫表雄甾酮亞硫酸基轉移酵素之同型二聚體及單體。此外，利用抗生素做大腸桿菌的菌株篩選及連續的親和性管柱純化策略，我們得到了同時具備酚亞硫酸基轉移酵素及脫氫表雄甾酮亞硫酸基轉移酵素之催化功能的異型二聚體。經由圓二色偏光光譜法來觀察各式聚合體於二級及三級結構上的差異，並經由酵素動力分析、受質抑制反應、與受質酵素結合能力的探討來幫助我們推論亞硫酸基轉移酵素之催化機制。此外，熱穩定性、酸鹼值耐受度實驗的分析，能幫助我們研究同型二聚體、單體、與異型二聚體在結構上的穩定性。經由對於同型二聚體、單體、與異型二聚體之各種特性的分析，我們能瞭解四級結構對於細胞質亞硫酸基轉移酵素的影響與重要性，更甚之，幫助我們推論藉由多體聚合化產生具有新功能蛋白質的可能性。

Sulfotransferase (SULT), mostly known as a homodimer in solution, belongs to an enzyme family that is responsible for the sulfonation of biomolecules which regulates many biological functions. In order to study the effects of quaternary structure on the activity of the enzyme, we prepared homodimer and monomer of SULT1A1 and SULT2A1 by site-directed mutagenesis and confirmed by size-exclusion liquid chromatography. Moreover, the bifunctional enzyme, SULT1A1/SULT2A1 heterodimer, was obtained from Escherichia coli by antibiotic selection and serially purified by affinity fusion tag. The secondary and tertiary structures of SULT in various combinations were investigated by circular dichroism spectrum. Enzyme kinetics, substrate inhibition and substrate binding affinity were also performed to deduce the SULT catalytic mechanism and actions. Thermal inactivation, pH tolerance, and conformational stabilities were determined by enzyme assay analysis. The above analyses toward homodimer, monomer, and heterodimer of SULT1A1 and SULT2A1, facilitate us to understand the importance of quaternary structure to cytosolic sulfotransferases, furthermore, assist us to understand how multimerization in proteins may produce novel biofunction.