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.

Abstract

The binding of two similar spin-labeled fatty acyl-CoA analogues, 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 (citrate oxaloacetate-lyase (pro-3S-CH2COO- leads to acetyl-CoA) EC 4.1.3.7) has been compared. The binding of the short chain analogue 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 analogue was complex and in excess of 2 mol/dimer. Competitive binding experiments using either analogue in the presence of various nucleotides and substrates revealed differences in the binding of the long and short chain analogues. 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 differing effects on the binding of the spin-labeled analogues. Oxalacetate was shown to decrease the binding of the long chain analogue but to have no effect on the binding of the short chain. This result was supported by kinetic measurements. The competitive binding experiments with the long chain analogue suggested that its complex isotherm resulted from binding in two classes of sites, i.e. two 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 two 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 (Remington, S., Wiegand, G., and Huber, R. (1982) J. Mol. Biol. 158, 111-152).