REGULATION OF ENZYMES BY FATTY ACYL COENZYME-A - INTERACTIONS OF SHORT AND LONG-CHAIN SPIN-LABELED ACYL-COA WITH THE ACETYL-COA SITE ON PIG-HEART CITRATE SYNTHASE
The binding of 2 similar spin-labeled fatty acyl-CoA analogs, one short chain [6-doxyloctanoyl-CoA (S-(2-(5-carboxybutyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyl-N-oxyl)-CoA)] and one long chain [6-doxylstearoyl-CoA (S-(2-(5-carboxybutyl)-2-dodecyl-4,4-dimethyl-3-oxazolidinyl-N-oxyl)-CoA)] to pig heart citrate synthase was compared. The binding of the short chain analog could be satisfactorily fit by a classical treatment (independent, noninteracting sites) with well defined stoichiometry: 2 mol of spin label bound per mol of dimeric enzyme. Binding of the long chain analog was complex and in excess of 2 mol/dimer. Competitive binding experiments using either analog in the presence of various nucleotides and substrates revealed differences in the binding of the long and short chain analogs. These additional studies, together with kinetic measurements, implied isosteric binding of acyl-CoA, ATP, NADPH, NADH, NADP, acetyl-CoA and partial isosteric binding of the long chain acyl-CoA. Binding of NADPH and NADP to the same form of the enzyme, perhaps through overlapping sites, was kinetically verified even though these nucleotides had different effects on the binding of the spin-labeled analogs. Oxalacetate decreased the binding of the long chain analog but had no effect on the binding of the short chain. This result was supported by kinetic measurements. The competitive binding experiments with the long chain analog suggested that its complex isotherm resulted from binding in 2 classes of sites, i.e., 2 cooperative nucleotide sites and other sites. An empirical mathematical model employing this rationale provided a satisfactory fit for the binding of fatty acyl-CoA to citrate synthase. A spin-labeled fatty acid which was not bound by the native enzyme was appreciably bound in the presence of additional palmitoyl-CoA. This binding might be identified with one of the 2 sets of binding sites proposed in the model. These and previous results on acyl-CoA binding were correlated with the properties of the CoA binding site defined crystallographically.