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 omega-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 PPi. The apparent Km values for ATP and CoA in the presence of the acyl matrix are similar to the respective Km values observed in the presence of the free acid substrate. The apparent Km for the acyl matrix is 10-fold higher (0.5 mM) than the apparent Km 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 omega-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.
D000818:Animals; D003066:Coenzyme A Ligases / Q000378:metabolism*; D003488:Cyanogen Bromide; D007202:Indicators and Reagents; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D013058:Mass Spectrometry; D011134:Polysaccharides / Q000138:chemical synthesis*; D051381:Rats; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D012685:Sepharose / Q000031:analogs & derivatives / Q000138:chemical synthesis*; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D007202:Indicators and Reagents; D011134:Polysaccharides; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C037384:stearate-sepharose; D012685:Sepharose; D003066:Coenzyme A Ligases; C090799:FAA2 protein, S cerevisiae; D000098859:Long-Chain-Fatty-Acid-CoA Ligase; D003488:Cyanogen Bromide