Facile enzymatic synthesis of fatty acylcoenzyme A thioesters.
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.
D000214:Acyl Coenzyme A / Q000138:chemical synthesis*; D000818:Animals; D003066:Coenzyme A Ligases / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005260:Female; D007700:Kinetics; D008722:Methods; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats
D000214:Acyl Coenzyme A; D004800:Enzymes, Immobilized; D003066:Coenzyme A Ligases