Regulatory Mechanisms Involving Nicotinamide Adenine Nucleotides as Allosteric Effectors: IV. PHYSICOCHEMICAL STUDY AND BINDING OF LIGANDS TO CITRATE SYNTHASE

Abstract

Citrate synthase (EC 4.1.3.7) from Escherichia coli is purified to homogeneity. The minimal molecular weight from amino acid analysis is about 62,000. The molecular weight of sodium dodecyl sulfate monomers is 60,000 to 65,000. Results of peptide fingerprinting suggest that the enzyme is composed of only one kind of subunit. The catalytically active protein is a tetramer with a molecular weight of 248,000. At a pH of 8.0 to 9.5, citrate synthase, both in dilute and concentrated solutions, is present as an equilibrium mixture of the catalytically inactive monomer and octamer and catalytically active tetramer. At a pH of 7.0 the equilibrium is shifted toward the formation of octamer and in the presence of dithiothreitol this is the only species present. At a pH of 11.0 the enzyme irreversibly dissociates to form monomers. Titration with thiol reagents shows that the native enzyme has 1.5 to 2 sulfhydryl groups per monomer which are readily available to the reagents. Enzyme denaturated by sodium dodecyl sulfate shows the presence of about eight sulfhydryl groups per monomer. The reactivity of the exposed sulfhydryl groups does not change in the presence of substrate, acetyl coenzyme A (acetyl-CoA), or the allosteric inhibitors, α-ketoglutarate and DPNH. Equilibrium-binding studies show that acetyl-CoA binds to the catalytically active tetramer and catalytically inactive octamer in a cooperative manner, both in the absence and presence of KCl. In initial velocity studies, however, the cooperative rate-concentration plots in the absence of KCl become Michaelian hyperbolas in its presence. It is surmised that KCl possibly affects only the rate constants associated with the binding of various reactants. The allosteric inhibitors, α-ketoglutarate and DPNH, bind to the enzyme in a negatively cooperative manner in the absence of acetyl-CoA but in a positively cooperative manner in its presence. The binding of both the inhibitors, especially that of DPNH, is considerably enhanced in the presence of the substrate. A quasi-ordered binding of inhibitors is, therefore, postulated during catalysis. All of the experimental observations can be rationalized if it is assumed that the various polymeric forms of the enzyme exist in the absence of ligands in a conformation which binds the substrates with poor affinity. The cooperativity of the binding plots is considered to arise by the differential binding of substrates and inhibitors to two states or conformations of the protein.