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Multiple active conformers of mouse ornithine decarboxylase
S E Tsirka1, C W Turck, P Coffino
1Department of Microbiology and Immunology, University of California San Francisco 94143.
The Biochemical Journal
|July 1, 1993
Summary
Denaturing and renaturing mouse ornithine decarboxylase (ODC) restored enzyme activity but altered its structure. This conformational change affected cysteine residue accessibility, impacting radioactive labeling with alpha-difluoromethylornithine (DFMO).
Area of Science:
- Biochemistry
- Enzymology
- Protein chemistry
Background:
- Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis.
- Understanding ODC's structural dynamics is crucial for drug development targeting related diseases.
Purpose of the Study:
- To investigate the effects of denaturation and renaturation on mouse ODC structure and function.
- To characterize the conformational changes and their impact on enzyme activity and substrate binding.
Main Methods:
- Purification of recombinant mouse ODC.
- Denaturation using urea and guanidinium chloride.
- Enzyme activity assays with ornithine and alpha-difluoromethylornithine (DFMO).
- Circular dichroism (c.d.) spectroscopy.
- Analysis of reactive cysteine residues.
Main Results:
- Enzymic activity was recovered after denaturation/renaturation.
- Kinetics for ornithine and DFMO decarboxylation remained unchanged.
- Renatured ODC showed reduced stable labeling with radioactive DFMO compared to native ODC.
- Native ODC had 4 reactive cysteines, while renatured ODC had 7.
Conclusions:
- Denaturation/renaturation induces a conformational change in ODC without altering substrate decarboxylation kinetics.
- The conformational change affects the accessibility or stability of cysteine residues, particularly Cys-360, influencing DFMO modification.
- These findings provide insights into ODC's structural flexibility and its implications for enzyme inhibition.