TY - JOUR
T1 - Substrate gating confers steroid specificity to estrogen sulfotransferase
AU - Petrotchenko, Evgeniy V.
AU - Doerflein, Mary E.
AU - Kakuta, Yoshimitsu
AU - Pedersen, Lars C.
AU - Negishi, Masahiko
PY - 1999/10/15
Y1 - 1999/10/15
N2 - Estrogen sulfotransferase (EST) exhibits a high substrate specificity and catalytic efficiency toward estrogens such as estradiol (E2) but insignificant ability to sulfate hydroxysteroids such as dehydroepiandrosterone (DHEA). To provide the structural basis for this estrogen specificity, we mutated amino acid residues that constitute the substrate-binding site of EST. Among these mutants, only Tyr-81 decreased E2 and increased DHEA sulfotransferase activities. Substitution for Tyr-81 by smaller hydrophobic residues increased K(m(E2)) for E2 activity, whereas the k(cat(E2)) remained relatively constant. The Y81L mutant exhibited the same DHEA activity as wild-type hydroxysteroid sulfotransferase, for which K(m(DHEA)) remained relatively constant, and k(cat(DHEA)) was markedly increased. The side chain of Tyr-81 is directed at the A-ring of the E2 molecule in the substrate-binding pocket of EST, constituting a steric gate with Phe-142 sandwiching E2 from the opposite side. The present mutagenesis study indicates that the 3β-hydroxyl group of the DHEA molecule is excluded from the catalytic site of EST through steric hindrance of Tyr-81 with the C- 19 methyl group of DHEA. Thus, this stricture-like gating caused by steric hindrance appears to be a structural principle for conferring estrogen specificity to EST.
AB - Estrogen sulfotransferase (EST) exhibits a high substrate specificity and catalytic efficiency toward estrogens such as estradiol (E2) but insignificant ability to sulfate hydroxysteroids such as dehydroepiandrosterone (DHEA). To provide the structural basis for this estrogen specificity, we mutated amino acid residues that constitute the substrate-binding site of EST. Among these mutants, only Tyr-81 decreased E2 and increased DHEA sulfotransferase activities. Substitution for Tyr-81 by smaller hydrophobic residues increased K(m(E2)) for E2 activity, whereas the k(cat(E2)) remained relatively constant. The Y81L mutant exhibited the same DHEA activity as wild-type hydroxysteroid sulfotransferase, for which K(m(DHEA)) remained relatively constant, and k(cat(DHEA)) was markedly increased. The side chain of Tyr-81 is directed at the A-ring of the E2 molecule in the substrate-binding pocket of EST, constituting a steric gate with Phe-142 sandwiching E2 from the opposite side. The present mutagenesis study indicates that the 3β-hydroxyl group of the DHEA molecule is excluded from the catalytic site of EST through steric hindrance of Tyr-81 with the C- 19 methyl group of DHEA. Thus, this stricture-like gating caused by steric hindrance appears to be a structural principle for conferring estrogen specificity to EST.
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U2 - 10.1074/jbc.274.42.30019
DO - 10.1074/jbc.274.42.30019
M3 - Article
C2 - 10514486
AN - SCOPUS:0033569893
VL - 274
SP - 30019
EP - 30022
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 42
ER -