Coenzyme binding by native and chemically modified pig heart triphosphopyridine nucleotide dependent isocitrate dehydrogenase.
The binding of TPNH to native and chemically modified pig heart TPN-dependent isocitrate dehydrogenase was studied by the techniques of ultrafiltration and fluorescence enhancement. A single site (per peptide chain) was found for TPNH with a dissociation constant (KD = 1.45 muM) that is quantitatively comparable to the Michaelis constant. The oxidized coenzyme, TPN+, weakens the binding of TPNH. The substrate manganous isocitrate also inhibits the binding of TPNH and, reciprocally, TPNH inhibits the binding of manganous isocitrate, suggesting that binding to the reduced coenzyme and substrate sites is mutually exclusive. Ultrafiltration experiments with carbonyl [14C]TPN+ revealed the existence of two sites with a dissociation constant (49 muM) more than ten times higher than the Michaelis constant. This observation excludes a random mechanism for isocitrate dehydrogenase or a sequential mechanism in which TPN+ binds first. Four chemically modified isocitrate dehydrogenases have been prepared: enzyme inactivated by reaction of a single methionyl residue with iodoacetate, by modification of a glutamyl residue by glycinamide (in the presence of a water soluble carbodiimide), by reaction of four cysteines successively with 5,5'-dithiobis(2-nitrobenzoic acid) and potassium cyanide, or by addition of two cysteine residues to N-ethylmaleimide. These enzymes were tested for their ability to bind TPN+, TPNH, and manganous isocitrate. In the cases of the cysteinyl and glutamyl-modified enzymes, inactivation appears to be due primarily to loss of the ability to bind the substrate manganous isocitrate. In constrast, the methionyl residue may participate in the coenzyme binding site or, more likely, may be involved in a step in catalysis subsequent to binding.
D000818:Animals; D001665:Binding Sites; D005033:Ethylmaleimide / Q000494:pharmacology; D007461:Iodoacetates; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology*; D009249:NADP; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013552:Swine; D013861:Thiocyanates; D014462:Ultrafiltration
D007461:Iodoacetates; D013861:Thiocyanates; D009249:NADP; D007521:Isocitrate Dehydrogenase; D005033:Ethylmaleimide