Interaction of a paramagnetic analogue of oxaloacetate with citrate synthase.
Electron paramagnetic resonance studies have indicated that nitrosodisulfonate binds to pig heart citrate synthase. Titration of the enzyme with nitrosodisulfonate revealed several binding sites for the probe per subunit with one site (KD approximately 0.1 mM) having a greater affinity than the others. The substrate, oxaloacetate, competed very effectively for one of the nitrosodisulfonate binding sites (KD less than 10(-2) mM) at the same time eliminating the weaker probe binding sites. Citrate and (R)- and (S)-malates also displaced the probe. Failure to resolve low- and high-field shoulder in the high gain--high modulation electron paramagnetic resonance spectra of the enzyme--nitrosodisulfonate system indicated that the bound probe was "weakly immobilized". However, the electron paramagnetic resonance spectrum of the bound probe changed to one typical of a "strongly immobilized" nitroxide upon the addition of a saturating concentration of the substrate acetyl coenzyme A (acetyl-CoA) to the enzyme--nitrosodisulfonate system, indicating the formation of a ternary acetyl-CoA-enzyme-probe complex. Titration of the acetyl-CoA saturated enzyme with the probe indicated one binding site per subunit (KD = 0.37 mM). Thus, nitrosodisulfonate may be considered as a paramagnetic analogue of oxaloacetate in its interaction with citrate synthase. These results are compared with our previous studies with this enzyme, employing a spin-labeled acyl coenzyme A (acyl-CoA) derivative [Weidman, S. W., Drysdale, G. R., & Mildvan, A. S. (1973) Biochemistry 12, 1874--1883].
D002950:Citrate (si)-Synthase; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D010071:Oxaloacetates; D007652:Oxo-Acid-Lyases; D011485:Protein Binding; D013113:Spin Labels
D010071:Oxaloacetates; D013113:Spin Labels; D002950:Citrate (si)-Synthase; D007652:Oxo-Acid-Lyases