title: Investigation of enzyme-substrate complexes by affinity chromatography. Application to pig heart citrate synthase.
abstract: The relative affinities of Sepharose gels, to which coenzyme A (CoA-SH) and CoA-SH analogues were bound through a well defined site, for citrate synthase were determined. The relative eluting power of coenzyme derivatives for the CoA-SH-gel and the Matrex Gel Blue-bound enzyme was measured, and the influence of oxaloacetate on the binding of the enzyme investigated. From the results, the contributions of different parts of the coenzyme to its binding in the active site and kinetic concepts are derived and found to be in complete agreement with corresponding data for citrate synthase obtained from kinetic measurements reported in the literature. It is demonstrated for some other CoA-SH-specific enzymes that affinity chromatography is of value as an additional tool for the comparative investigation of binding sites of enzymes which depend on the same coenzyme.
mesh_terms: D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods; D002950:Citrate (si)-Synthase / Q000032:analysis*; D003065:Coenzyme A / Q000032:analysis; D003067:Coenzymes / Q000032:analysis; D009206:Myocardium / Q000201:enzymology*; D010071:Oxaloacetates; D007652:Oxo-Acid-Lyases / Q000032:analysis*; D011506:Proteins / Q000378:metabolism; D011685:Purine Nucleotides; D011742:Pyrimidine Nucleotides; D013237:Stereoisomerism; D013552:Swine
chemical_list: D003067:Coenzymes; D010071:Oxaloacetates; D011506:Proteins; D011685:Purine Nucleotides; D011742:Pyrimidine Nucleotides; D002950:Citrate (si)-Synthase; D007652:Oxo-Acid-Lyases; D003065:Coenzyme A