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Functional activity of 3beta-hydroxysteroid dehydrogenase/isomerase
J I Mason1, D Naville, B W Evans
1Department of Clinical Biochemistry, Royal Infirmary of Edinburgh NHS Trust, United Kingdom.
Endocrine Research
|January 15, 1999
Summary
The human 3beta-hydroxysteroid dehydrogenase/steroid delta5-isomerase (3beta-HSD/isomerase) enzyme
Area of Science:
- Biochemistry
- Enzymology
- Steroid Metabolism
Background:
- 3beta-hydroxysteroid dehydrogenase/steroid delta5-isomerase (3beta-HSD/isomerase) is crucial for steroid hormone biosynthesis.
- Understanding its catalytic mechanism is key to comprehending steroidogenic pathways.
Purpose of the Study:
- To investigate the role of Tyrosine 253 (Tyr253) in the isomerase activity of human 3beta-HSD/isomerase.
- To elucidate the coenzyme activation mechanism and enzyme conformational changes.
Main Methods:
- Baculovirus expression of wild-type and Y253F mutant human 3beta-HSD/isomerase in Sf9 insect cells.
- Protein purification and characterization.
- UV spectral analyses using a competitive isomerase inhibitor (19-nortestosterone).
Main Results:
- Wild-type 3beta-HSD/isomerase, but not the Y253F mutant, induced spectral changes in 19-nortestosterone upon coenzyme activation.
- NADH activation resulted in a greater spectral shift than NAD+.
- Tyr253 is essential for the isomerase reaction, likely acting as a general acid (proton donor).
Conclusions:
- Direct evidence implicates Tyr253 as the general acid in the 3beta-HSD/isomerase mechanism.
- Coenzyme activation profiles support a two-step mechanism where NADH-induced 3beta-HSD activity promotes the isomerase conformation.