利用人類脫氫表雄甾酮亞硫酸基轉移酵素之序列與結構比對探討其催化機制與功能

Abstract

在此研究當中，主要利用人類脫氫表雄甾酮亞硫酸基轉移酵素之胺基酸序列與已知結晶結構來探討其催化機制與功能。亞硫酸基轉移酵素目前大部分已知為同型二聚體，在生理上主要將生物分子亞硫酸化，進而調控許多生理反應與功能。在此研究中，首先為了要瞭解四級結構對此酵素活性之影響，因而藉由定點突變法製備單體之人類脫氫表雄甾酮亞硫酸基轉移酵素與經由膠體過濾分析，我們成功得到單體的亞硫酸基轉移酵素。進而藉由圓二色偏光光譜法來觀測單體與雙體在二級與三級結構上之差異。另外，利用酵素動力學分析探討其催化機制，受質抑制作用，與受質結合能力分析。此外，藉由熱穩定度與尿素變性分析單體與雙體在構型上的穩定度。另一方面，亞硫酸基轉移酵素共有一個特徵：受質抑制反應。藉由交叉比對分析其結晶結構之二體與三體錯合物，推測可能調控受質抑制反應與受質位向之胺基酸，進而探討受質抑制反應的起源與機制。在此研究中，我們成功地瞭解受質抑制反應與受質位向之間相關性。此外，利用定點突變法與其進一步分析指出，此胺基酸可能在其他亞硫酸基轉移酵素中之相對位置對於受質抑制反應扮演相類似的調控角色。

In this research we deduce the human dehydroepiandrosterone sulfotransferase (SULT2A1) catalytic mechanism and function by the comparison of its amino acid sequences and spatially resolved crystal structures. Sulfotransferase (SULT), mostly known to be homodimer in solution, belongs to an enzyme family that is responsible for the sulfonation of biomolecules and then the regulation of many biological functions. In order to study the effect of quaternary structure on the activity of the enzyme, SULT2A1 monomer mutant is prepared by site-directed mutagenesis and confirmed by gel filtration. Further, the differences of secondary and tertiary structures between dimer and monomer, respectively, are investigated by circular dichroism spectrum. Enzyme kinetics, substrate inhibition and substrate binding affinity are facilitated to deduce the catalytic mechanism and actions. Thermal inactivation and conformational stabilities of dimer and monomer are also determined by enzyme assay and urea unfolding analysis individually. Another general feature of sulfotransferase is substrate inhibition. Following the analysis of the structures of binary and ternary complexes of sulfotransferase, we are able to elucidate the specific amino acids responsible for substrate inhibition and binding orientations. And then we propose the origin and mechanism of substrate inhibition and uncover the relationship between the substrate inhibition and binding orientations. Site-directed mutagenesis and resulting analysis confirm that the involvement of specific amino acid residue in substrate inhibition may be a general feature in all cytosolic sulfotransferases.