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Specific acetylation of essential lysine residues in malonyl-CoA decarboxylase
Abstract:
1. Malonyl-CoA decarboxylase from the uropygial gland of goose was inactivated by treatment with low concentrations of acetic anhydride in Tris-acetate buffer. 2. The degree of inactivation depended on the concentration of the reagent and time of incubation. 3. Treatment of the enzyme with [1-14C] acetic anhydride resulted in covalent attachment of the label exclusively to the decarboxylase protomer. 4. Evidence is presented that the label incorporated into the protein was exclusively in N-epsilon-acetyllysine. Graphical analysis suggested that acetylation of about two lysine residues/subunit would be required for complete inactivation of the enzyme. 5. Malonyl-CoA, acetyl-CoA, propionyl-CoA and methylmalonyl-CoA afforded partial protection to the enzyme against inactivation by acetic anhydride. 6. 3'-Dephosphomalonyl-CoA was found to be an alternate substrate for the enzyme with approximately the same Km and V values as for malonyl-CoA. 7. This alternate substrate also partially protected the enzyme from inactivation, whereas adenine nucleotides with 2'-, 3', or 5'-phosphates did not protect the enzyme. 8. These results suggest that a lysine residue acetylated by acetic anhydride is essential for the enzyme activity and that it is probably at or near the active site. The results are consistent with the suggestion that the enzyme is composed of four identical subunits.
Insights
Goose malonyl-CoA decarboxylase inactivation by acetic anhydride reveals essential lysine residues at the active site. This study identifies a critical lysine for enzyme function, crucial for understanding enzyme mechanisms.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Protein modification
Background:
- Malonyl-CoA decarboxylase (MCD) is a key enzyme in fatty acid metabolism.
- Understanding the catalytic mechanism and active site of MCD is crucial for metabolic research.
Purpose of the Study:
- To investigate the inactivation mechanism of goose uropygial gland MCD using acetic anhydride.
- To identify the specific amino acid residues involved in enzyme activity.
Main Methods:
- Enzyme inactivation assays with varying acetic anhydride concentrations and incubation times.
- Radioactive labeling with [1-14C] acetic anhydride to identify covalent modification sites.
- Analysis of substrate protection against inactivation.
Main Results:
- Acetic anhydride irreversibly inactivated MCD, with inactivation dependent on reagent concentration and time.
- Labeling occurred exclusively on the decarboxylase protomer, specifically at N-epsilon-acetyllysine residues.
- Acetylation of approximately two lysine residues per subunit was required for complete inactivation.
- Malonyl-CoA, acetyl-CoA, propionyl-CoA, methylmalonyl-CoA, and 3'-dephosphomalonyl-CoA partially protected the enzyme.
Conclusions:
- A specific lysine residue, likely at or near the active site, is essential for goose MCD activity.
- The enzyme is likely composed of four identical subunits, based on graphical analysis.
- These findings provide insights into the catalytic mechanism and active site structure of malonyl-CoA decarboxylase.