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Substrate gating confers steroid specificity to estrogen sulfotransferase
E V Petrotchenko1, M E Doerflein, Y Kakuta
1Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
The Journal of Biological Chemistry
|October 9, 1999
Summary
Estrogen sulfotransferase (EST) shows high specificity for estrogens like estradiol (E2). Mutating Tyr-81 revealed its crucial role in EST's substrate specificity, controlling access to the active site.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Estrogen sulfotransferase (EST) is crucial for steroid hormone metabolism.
- EST exhibits high specificity for estrogens (e.g., estradiol, E2) but low activity towards other hydroxysteroids (e.g., dehydroepiandrosterone, DHEA).
- Understanding the structural basis of EST's substrate specificity is essential for enzyme engineering and drug development.
Purpose of the Study:
- To elucidate the structural determinants responsible for estrogen specificity in EST.
- To investigate the role of specific amino acid residues in the substrate-binding site of EST.
- To provide a structural explanation for why EST efficiently sulfates estrogens but not DHEA.
Main Methods:
- Site-directed mutagenesis of amino acid residues within the EST substrate-binding site.
- Enzymatic assays to measure sulfotransferase activity towards E2 and DHEA for wild-type and mutant EST.
- Analysis of kinetic parameters (Km and kcat) to quantify changes in substrate affinity and catalytic efficiency.
- Structural analysis of the EST substrate-binding pocket and interactions with E2.
Main Results:
- Mutation of Tyr-81 significantly altered EST activity, decreasing E2 sulfation and increasing DHEA sulfation.
- Substitution of Tyr-81 with smaller hydrophobic residues increased the Km for E2, indicating reduced affinity.
- The Y81L mutant showed significantly increased kcat for DHEA sulfation, approaching wild-type hydroxysteroid sulfotransferase activity.
- Structural analysis revealed Tyr-81 forms a steric gate with Phe-142, controlling substrate entry into the active site.
Conclusions:
- Tyr-81 acts as a critical steric gate, conferring estrogen specificity to EST.
- The steric hindrance between Tyr-81 and the C-19 methyl group of DHEA prevents its efficient binding and sulfation.
- This structural mechanism explains the selective sulfation of estrogens over other hydroxysteroids by EST.