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Structure-function studies of human 5-alpha reductase type 2 using site directed mutagenesis.
F O Baxter1, S Trivic, I R Lee
1School of Biomedical Sciences, Curtin University of Technology, GPO Box U1987, Perth, WA 6845, Australia.
Summary
Site-directed mutagenesis identified key residues in human steroid 5-alpha-reductase isoenzymes. Specific amino acid substitutions in steroid 5-alpha-reductase type 1 (5AR1) and type 2 (5AR2) impact inhibitor binding and substrate affinity.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzyme Kinetics
Background:
- Human steroid 5-alpha-reductase isoenzymes (5AR1 and 5AR2) are critical drug targets.
- Understanding inhibitor and substrate binding is essential for drug development.
Purpose of the Study:
- To identify specific amino acid residues in 5AR1 and 5AR2 involved in inhibitor and substrate binding.
- To elucidate the differential roles of specific residue regions in enzyme activity and inhibition.
Main Methods:
- Site-directed mutagenesis was employed to create chimeric and substituted 5AR1 and 5AR2 enzymes.
- Enzyme kinetics were analyzed by measuring Km for testosterone and Ki for Finasteride.
Main Results:
- Replacing 5AR1 residues 26-29 (AVFA) with 5AR2's 21-24 (GALA) decreased Finasteride resistance, indicating AVFA's role in binding.
- Substitution of 5AR2 residues 15-17 (ATL) with 5AR1's QCA significantly decreased Km and increased Finasteride resistance, highlighting ATL's importance.
Conclusions:
- Residues 26-29 of 5AR1 and residues 15-17 of 5AR2 are crucial for inhibitor and substrate binding.
- Residues 21-24 of 5AR2 and residues 18-20 of 5AR1 do not appear to directly participate in binding interactions.