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Malonyl coenzyme A synthetase. Purification and properties.
The Journal of Biological Chemistry
|April 25, 1985
Summary
This study reports the first purification of malonyl coenzyme A synthetase from Pseudomonas fluorescens. The enzyme
Area of Science:
- Biochemistry
- Enzymology
- Microbial Metabolism
Background:
- Malonyl coenzyme A synthetase (EC 6.2.1.14) plays a role in microbial metabolic pathways.
- Enzyme induction in Pseudomonas fluorescens grown on malonate as a sole carbon source suggests specific metabolic adaptation.
Purpose of the Study:
- To purify and characterize malonyl coenzyme A synthetase from Pseudomonas fluorescens.
- To elucidate the enzyme's kinetic properties and cofactor requirements.
Main Methods:
- Enzyme purification using ammonium sulfate precipitation, Sephadex G-150 gel filtration, DEAE-Sephacel ion exchange, and hydroxylapatite chromatography.
- Electrophoretic analysis for homogeneity.
- Determination of molecular weight, optimal pH, and kinetic parameters (Km, Vmax).
Main Results:
- Achieved over 30-fold purification of electrophoretically homogeneous malonyl coenzyme A synthetase.
- Determined enzyme molecular weight (98,000 Da, two subunits), optimal pH (7.5), and requirement for ATP, CoA, and Mg2+.
- Calculated kinetic parameters (Km, Vmax) for malonate, ATP, and CoA, and demonstrated immunogenicity of the purified enzyme.
Conclusions:
- Successfully purified and characterized malonyl coenzyme A synthetase, providing insights into its properties.
- The enzyme's activity is dependent on specific substrates and cofactors, with Michaelis-Menten kinetics observed.
- The generated antiserum can inhibit enzyme activity, useful for further immunological studies.