The interactions of adenylates with allosteric citrate synthase.
Evidence is presented that a number of derivatives of adenylic acid may bind to the allosteric NADH binding site of Escherichia coli citrate synthase. This evidence includes the facts that all the adenylates inhibit NADH binding in a competitive manner and that those which have been tested protect an enzyme sulfhydryl group from reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) in the same way that NADH does. However, whereas NADH is a potent inhibitor of citrate synthase, most of the adenylates are activators. The best activator, ADP-ribose, increases the affinity of the enzyme for the substrate, acetyl-CoA, and saturates the enzyme in a sigmoid manner. A fluorescence technique, involving the displacement of 8-anilino-1-naphthalenesulfonate from its complex with citrate synthase, is used to obtain saturation curves for several nucleotides under nonassay conditions. It is found that acetyl-coenzyme A, coenzyme A, and ADP-ribose all bind to the enzyme cooperatively, and that the binding of each becomes tighter in the presence of KCl, the activator, and oxaloacetic acid (OAA), the second substrate. Another inhibitor, alpha-ketoglutarate, can complete with OAA in the absence of KCl but not in its presence. The nature of the allosteric site of citrate synthase, and the modes of action of several activators and inhibitors, are discussed in the light of this evidence.
D000227:Adenine Nucleotides / Q000494:pharmacology*; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D000495:Allosteric Site; D002950:Citrate (si)-Synthase / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000494:pharmacology; D007652:Oxo-Acid-Lyases / Q000378:metabolism*; D011485:Protein Binding
D000227:Adenine Nucleotides; D009243:NAD; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D002950:Citrate (si)-Synthase; D007652:Oxo-Acid-Lyases