Conversion of the Nucleotide Specificity of Phenol Sulfotransferase: Modifying the Binding Pocket of 3’-Phosphate of PAP by Site-Directed Mutagenesis

• Main Authors: Yu-Shan Hsiao, 蕭于珊
• Other Authors: Yuh-Shyong Yang
• Format: Others
• Language: en_US
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/13827462192426874439

碩士 === 國立交通大學 === 生物科技研究所 === 89 === 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.