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Essential tyrosine residues in 3-ketosteroid-delta(1)-dehydrogenase from Rhodococcus rhodochrous
1Department of Chemistry, Faculty of Science, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa, 920-1192, Japan.
Journal of Biochemistry
|September 30, 1999
Summary
Tetranitromethane treatment inactivated Rhodococcus rhodochrous 3-ketosteroid-Delta(1)-dehydrogenase by nitrating tyrosine-116. Mutations revealed tyrosine-121 is crucial for catalysis, while tyrosines-104 and -116 aid substrate binding.
Area of Science:
- Biochemistry
- Enzymology
- Protein Chemistry
Background:
- 3-ketosteroid-Delta(1)-dehydrogenase from Rhodococcus rhodochrous is vital for steroid metabolism.
- Understanding enzyme active site residues is key to elucidating catalytic mechanisms.
Purpose of the Study:
- To investigate the role of specific tyrosine residues in the catalytic activity and substrate binding of 3-ketosteroid-Delta(1)-dehydrogenase.
- To identify the site of tetranitromethane modification in the enzyme.
Main Methods:
- Enzyme treatment with tetranitromethane and subsequent peptide analysis.
- Site-directed mutagenesis of tyrosine residues (Y-104, Y-116, Y-121).
- Enzyme kinetics assays (K(m), K(d)) and spectrophotometric analysis of wild-type and mutant enzymes.
Main Results:
- Tetranitromethane selectively nitrated tyrosine-116, leading to loss of catalytic activity.
- Mutations at Y-104, Y-116, and Y-121 significantly altered substrate binding (K(m) and K(d) values increased).
- The Y-121 mutants showed drastically reduced catalytic efficiency, while Y-104 and Y-116 mutants primarily affected substrate binding.
Conclusions:
- Tyrosine-121 is essential for the catalytic function of 3-ketosteroid-Delta(1)-dehydrogenase.
- Tyrosine-116 and Tyrosine-104 play critical roles in substrate steroid binding.
- Tetranitromethane is a useful reagent for probing tyrosine residue function in enzymes.