Purification and properties of benzoate-coenzyme a ligase, a Rhodopseudomonas palustris enzyme involved in the anaerobic degradation of benzoate
A soluble benzoate-coenzyme A (CoA) ligase was purified from the phototrophic bacterium Rhodopseudomonas palustris. Synthesis of the enzyme was induced when cells were grown anaerobically in light with benzoate as the sole carbon source. Purification by chromatography successively on hydroxylapatite, phenyl-Sepharose, and hydroxylapatite yielded an electrophoretically homogeneous enzyme preparation with a specific activity of 25 ..mu..mol/min per mg of protein and a molecular weight of 60,000. The purified enzyme was insensitive to oxygen and catalyzed the Mg/sup 2 +/ ATP-dependent formation of acyl-CoA from carboxylate and free reduced CoA, with high specificity for benzoate and 2-fluorobenzoate. Apparent K/sub m/ values of 0.6 to 2 ..mu..M for benzoate, 2 to 3 ..mu..M for ATP, and 90 to 120 ..mu..M for reduced CoA were determined. The reaction product, benzoyl-CoA, was an effective inhibitor of the ligase reaction. The kinetic properties of the enzyme match the kinetics of substrate uptake by whole cells and confirm a role for benzoate-CoA ligase in maintaining entry of benzoate into cells as well as in catalyzing the first step in the anaerobic degradation of benzoate by R. palustris.
- Research Organization:
- Cornell Univ., Ithaca, NY (USA)
- DOE Contract Number:
- FG02-86ER13495
- OSTI ID:
- 7164806
- Journal Information:
- J. Bacteriol.; (United States), Vol. 170:4
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
BENZOIC ACID
ANAEROBIC DIGESTION
LIGASES
ENZYME ACTIVITY
RHODOPSEUDOMONAS
ESTERS
PURIFICATION
BACTERIA
BIOCONVERSION
CARBOXYLIC ACIDS
DIGESTION
ENZYMES
MANAGEMENT
MICROORGANISMS
MONOCARBOXYLIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PROCESSING
WASTE MANAGEMENT
WASTE PROCESSING
550200* - Biochemistry