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Decarboxylation of alpha-difluoromethylornithine by ornithine decarboxylase
Abstract:
The mechanism of inactivation of rodent ornithine decarboxylase by alpha-difluoromethylornithine (DFMO) was studied using the inhibitor labelled with 14C in both the 1 and the 5 positions. [1-14C]DFMO was a substrate and was decarboxylated by the enzyme yielding 14CO2. A radioactive metabolite derived from [5-14C]DFMO was bound to the enzyme, and the extent of binding paralleled the irreversible inactivation of ornithine decarboxylase. The partition ratio of decarboxylation to binding was approx. 3.3. These results provide support for the postulated mechanism of action of DFMO [Metcalf, Bey, Danzin, Jung, Casera & Vevert (1978) J. Am. Chem. Soc. 100, 2551-2553], in which enzymic decarboxylation of the inhibitor leads to the generation of a conjugated imine, which then alkylates a nucleophilic residue on the enzyme.
Insights
Alpha-difluoromethylornithine (DFMO) inactivates rodent ornithine decarboxylase by forming a bound metabolite. This metabolite formation, linked to enzyme decarboxylation, explains DFMO's mechanism of action.
Area of Science:
- Biochemistry
- Enzymology
- Pharmacology
Background:
- Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis.
- Alpha-difluoromethylornithine (DFMO) is a known inhibitor of ODC.
- The precise mechanism of ODC inactivation by DFMO requires further elucidation.
Purpose of the Study:
- To investigate the mechanism of ornithine decarboxylase (ODC) inactivation by alpha-difluoromethylornithine (DFMO).
- To elucidate the role of inhibitor metabolism in ODC inactivation.
Main Methods:
- Utilized 14C-labeled alpha-difluoromethylornithine (DFMO) at the 1 and 5 positions.
- Studied the enzymatic decarboxylation of [1-14C]DFMO.
- Analyzed the binding of a metabolite derived from [5-14C]DFMO to the enzyme.
Main Results:
- [1-14C]DFMO was decarboxylated by ornithine decarboxylase, producing 14CO2.
- A radioactive metabolite from [5-14C]DFMO irreversibly bound to ornithine decarboxylase.
- The extent of metabolite binding correlated with enzyme inactivation.
- The partition ratio of decarboxylation to binding was approximately 3.3.
Conclusions:
- Enzymatic decarboxylation of DFMO generates a reactive intermediate.
- This intermediate forms a covalent adduct with ornithine decarboxylase, leading to irreversible inactivation.
- The findings support the proposed mechanism of DFMO action involving a conjugated imine intermediate.