TY - JOUR
T1 - Site-directed mutagenesis of the substrate-binding cleft of human estrogen sulfotransferase
AU - Hempel, Nadine
AU - Barnett, Amanda C.
AU - Bolton-Grob, Robyn M.
AU - Liyou, Nancy E.
AU - McManus, Michael E.
N1 - Funding Information:
This research was supported by the National Health and Medical Research Council of Australia. Helpful discussions with Dr. Ronald G. Duggleby, Dr. Lulu Brix, and Dr. Lisa Bidwell are gratefully acknowledged.
PY - 2000/9/16
Y1 - 2000/9/16
N2 - 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 characteristics of humSULT1E1, we have carried out site-directed mutagenesis of critical amino acids found in the substrate-binding cleft. Three single amino acid mutations of humSULT1E1 (V145E, H107A, and K85A) were created in this study. Kinetic studies were used to provide information about the importance of these residues in substrate specificity and catalysis, using a variety of substrates. Lysine at position 85 has been proposed to be within hydrogen bonding distance to the 3α-phenol group of β-estradiol, thereby stabilising the substrate in the active site. However, substitution to a neutral alanine at this position improved substrate specificity of humSULT1E1 for β-estradiol, estrone, and dehydroepiandrosterone (DHEA). The exchange of valine 145 for negatively charged glutamic acid markedly improved the ability of humSULT1E1 to sulfonate dopamine, but caused a reduction in specificity constants toward steroids tested, in particular DHEA. The presence of a histidine residue at position 107 was shown to be essential for the production of a functional protein, as substitution of this amino acid to alanine resulted in complete loss of activity of humSULT1E1 towards all substrates tested. (C) 2000 Academic Press.
AB - 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 characteristics of humSULT1E1, we have carried out site-directed mutagenesis of critical amino acids found in the substrate-binding cleft. Three single amino acid mutations of humSULT1E1 (V145E, H107A, and K85A) were created in this study. Kinetic studies were used to provide information about the importance of these residues in substrate specificity and catalysis, using a variety of substrates. Lysine at position 85 has been proposed to be within hydrogen bonding distance to the 3α-phenol group of β-estradiol, thereby stabilising the substrate in the active site. However, substitution to a neutral alanine at this position improved substrate specificity of humSULT1E1 for β-estradiol, estrone, and dehydroepiandrosterone (DHEA). The exchange of valine 145 for negatively charged glutamic acid markedly improved the ability of humSULT1E1 to sulfonate dopamine, but caused a reduction in specificity constants toward steroids tested, in particular DHEA. The presence of a histidine residue at position 107 was shown to be essential for the production of a functional protein, as substitution of this amino acid to alanine resulted in complete loss of activity of humSULT1E1 towards all substrates tested. (C) 2000 Academic Press.
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U2 - 10.1006/bbrc.2000.3473
DO - 10.1006/bbrc.2000.3473
M3 - Article
C2 - 11006110
AN - SCOPUS:0034675279
SN - 0006-291X
VL - 276
SP - 224
EP - 230
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 1
ER -