Facile enzymatic synthesis of fatty acylcoenzyme A thioesters

• Published in: Journal of lipid research 1982-12, Vol.23 (9), p.1368-1373
• Main Authors: Merrill, Jr, A H, Gidwitz, S, Bell, R M
• Format: Article
• Language: English
• Subjects: Acyl Coenzyme A - chemical synthesis; acyl-CoA synthetase; Animals; Coenzyme A Ligases - metabolism; Enzymes, Immobilized - metabolism; fatty acyl-CoA thioesters; Female; immobilized enzymes; Kinetics; liver; Methods; microsomes; Microsomes, Liver - enzymology; Rats
• ISSN: 0022-2275
• DOI: 10.1016/S0022-2275(20)38043-3

The fatty acid:CoA ligase (acyl-CoA synthetase, EC 6.2.1.3) of rat liver microsomes was solubilized with Triton X-100 and bound to Matrex Gel Red A. Fatty acid:CoA ligase immobilized on Matrex Gel Red A was active and proved useful for the synthesis of fatty acyl-CoA thioesters. The immobilized activity was characterized by a 3-fold higher apparent Km for ATP than the soluble activity, similar apparent Km values for CoA and palmitate, and a shift in the pH dependence. Quantitative incorporation of fatty acid or CoA was possible. Long chain-fatty acyl CoA thioesters were purified in a single step by hydrophobic chromatography on Octyl-Sepharose. In addition to producing the thioesters of typical fatty acids (e.g., myristic, palmitic, stearic, oleic, cis-vaccenic, linoleic, and arachidonic), analogs such as the fluorescent molecules beta-parinaroyl-CoA and palmitoyl-(1-N6-etheno-)CoA were easily synthesized. These procedures should be generally applicable for both the small-scale, i.e., 1 to 10 mumoles, and large-scale, i.e., 50 to 250 mumoles, scale preparation of numerous fatty acyl-CoA's and related compounds.