Site-directed mutagenesis of the substrate-binding cleft of human estrogensulfotransferase

The sulfonation of estrogens by human estrogen sulfotransferase (humSULT1E1 ) plays a vital role in controlling the active levels of these hormones in the body. To understand more fully the structural and functional characteri stics of humSULT1E1, we have carried out site-directed mutagenesis of criti cal amino acids found in the substrate-binding cleft. Three single amino ac id mutations of humSULT1E1 (V145E, H107A, and K85A) were created in this st udy. Kinetic studies were used to provide information about the importance of these residues in substrate specificity and catalysis, using a variety o f substrates, Lysine at position 85 has been proposed to be within hydrogen bonding distance to the 3 alpha-phenol group of beta-estradiol, thereby st abilising the substrate in the active site. However, substitution to a neut ral alanine at this position improved substrate specificity of humSULT1E1 f or beta-estradiol, estrone, and dehydroepiandrosterone (DHEA), The exchange of valine 145 for negatively charged glutamic acid markedly improved the a bility of humSULT1E1 to sulfonate dopamine, but caused a reduction in speci ficity constants toward steroids tested, in particular DHEA. The presence o f a histidine residue at position 107 was shown to be essential for the pro duction of a functional protein, as substitution of this amino acid to alan ine resulted in complete loss of activity of humSULT1E1 towards all substra tes tested, (C) 2000 Academic Press.