Sepharose-stearate as substrate for rat liver long-chain fatty acyl-CoA synthetase

Stearic acid coupled covalently to Sepharose 6B serves as substrate for thioesterification catalyzed by rat liver long-chain fatty acyl-CoA synthetase (ATP-forming) (EC 6.2.1.3). Availability as substrate is dependent upon the conservation of the free ω-terminal in addition to that of the free carboxyl function. The enzymatic overall formation of matrix-acyl-CoA in the presence of ATP and CoA as cosubstrates conforms to the stoichiometry reported for thioesterification of the free long-chain fatty acyl substrate. The preformed matrix-acyl-CoA serves as substrate for the backward synthetase reaction in the presence of AMP and PP_i- The apparent K_m values for ATP and CoA in the presence of the acyl matrix are similar to the respective K_m values observed in the presence of the free acid substrate. The apparent K_m for the acyl matrix is 10-fold higher (0.5 mM) than the apparent K_m value for the free acid. The feasibility of enzymatic thioesterification of bound long-chain fatty acids implies that the exact nature of the bulky chain situated between the carboxy and ω-terminal plays a secondary role in defining the fatty acyl substrate specificity for long-chain fatty acyl-CoA synthetase. Also, dissociation of bound long-chain fatty acids does not constitute an obligatory preliminary step to fatty acid thioesterification.