Purification and characterization of a medium chain acyl-coenzyme A synthetase.

ABSTRACT

Glycine conjugation is an important route of detoxification of many xenobiotic and endogenous carboxylic acids. A medium chain acyl-coenzyme A synthetase that catalyzes the first reaction of glycine conjugation was purified from bovine liver mitochondria by chromatographies on anion exchange, hydroxylapatite, affinity, and finally by gel filtration. The purified enzyme not only conjugates medium chain fatty acids, but also aromatic and arylacetic acids. The highest activity was shown with hexanoic acid. High activities were observed for benzoic acid derivatives with large alkyl and alkoxyl groups in the para- or meta-positions of the benzene ring. Ortho-substituted derivatives exhibited no activity. The enzyme was inhibited by iodoacetamide and salicylic acid, and activated by albumin. Salicylic acid was a competitive inhibitor of the enzyme, with an apparent Ki value of 37 microM. Enzyme activity increased 74% when the pH was raised from 7 to 10. Molecular weight of the purified medium chain acyl-coenzyme A synthetase was 65.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.