酚亞硫酸基轉移酵素核甘酸特異性之轉換: 利用定點突變修飾探討PAP 3’-Phosphate與酵素之結合區域

Abstract

亞硫酸基轉移酵素(sulfotransferase)的功能主要是催化所有已知的生物亞硫酸基轉移反應，此類酵素是以一個稱做3’-phosphoadenosine 5’-phosphosulfate (PAPS)的核甘酸為亞硫酸基提供者，催化亞硫酸基轉移至生物系統中各種親和受體的反應。有數種其他的核甘酸皆被發現可以當作酚亞硫酸基轉移酵素(phenol sulfotransferase, PST)於催化反應中之受質或輔因子，但唯其具有較低之催化反應效率 (Lin & Yang, 2000)。在此研究中，我們藉由一些細胞內亞硫酸基轉移酵素之單晶繞射結構與胺基酸序列的比較結果，找出了六個可能對於酵素與不同之核甘酸結合、催化特異性有關係的胺基酸，並利用定點突變的方法，將此六個胺基酸：Arg-253、Lys-254、Gly-255、Ser-134、Arg-126和Lys-44分別進行突變。透過對酚亞硫酸基轉移酵素突變株的動力學分析，我們發現在酵素與3’-phosphoadenosine 5’-phosphate (PAP)之3’-phosphate結合區域中，胺基酸Ser-134對於酵素受質PAP特異性之選擇上，扮演一關鍵性的角色；反之，其對於另一核甘酸Adenosine 5’-monophosphate (AMP)則沒有太大之影響。另外Ser-134在核甘酸與酵素的結合之影響亦遠大於對催化反應之影響。而其中兩個突變株酵素：S134E與S134R所表現之受質選擇性亦被改變成AMP而非原本野生株酵素之PAP。另外，動力學分析的結果發現，我們可以藉由單一之定點突變株酵素，使不同核甘酸對酵素之催化反應效率比值降低了十萬倍之多，並經此分析不同核甘酸對酵素反應特異性之轉換機制。

Sulfotransferase (ST) catalyzes all the known biological sulfonation, which involves the transfer of a sulfuryl group from a common sulfonate donor, 3’-phosphoadenosine 5’-phosphosulfate (PAPS), to a nucleophilic acceptor. Other nucleotides are also found to serve as cofactor or substrate of phenol sulfotransferase (PST) but with much less catalytic efficiency (Lin & Yang, 2000). We use site-directed mutagenesis to identify amino acid residues that affect the specificity of PST. Six amino acid residues are chosen for mutation based on the crystal structures and sequence comparison of several cytosolic and membrane STs. We analyze these mutants systematically and find that Ser-134 is important for the nucleotide specificity of 3’-phosphoadenosine 5’-phosphate (PAP) but not for adenosine 5’-monophosphate (AMP). The kinetic constants also indicate that this serine residue play a key role in nucleotide binding but not in catalysis. Two single mutants, S134E and S134R, are shown to prefer AMP rather PAP as cofactor. The ratio of catalytic efficiency of PST is altered for over five orders of magnitude with a single mutation. The mechanism for this switch of specificity is proposed.