Cyanate modification of essential lysyl residues of the diphosphopyridine nucleotide-specific isocitrate dehydrogenase of pig heart.
The DPN-specific isocitrate dehydrogenase of pig heart is totally and irreversibly inactivated by 0.05 M potassium cyanate at pH 7.4 A plot of the rate constant versus cyanate concentration is not linear, but rather exhibits saturation kinetics, implying that cyanate may bind to the enzyme to give an enzyme-cyanate complex (K equal 0.125 M) prior to the covalent reaction. In the presence of manganous ion the addition of isocitrate protects the enzyme against cyanate inactivation, indicating that chemical modification occurs in the active site region of the enzyme. The dependence of the decrease of the rate constant for inactivation on the isocitrate concentration yields a dissociation constant for the enzyme-manganese-isocitrate complex which agrees with the Michaelis constant. The allosteric activator ADP, which lowers the Michaelis constant for isocitrate, does not itself significantly affect the cyanate reaction; however, it strikingly enhances the protection by isocitrate. The addition of the chelator EDTA essentially prevents protection by isocitrate and manganous ion, demonstrating the importance of the metal ion in this process. The substrate alpha-ketoglutarate and the coenzymes DPN and DPNH do not significantly affect the rate of modification of the enzymes by cyanate. Incubation of isocitrate dehydrogenase with 14C-labeled potassium cyanate leads to the incorporation of approximately 1 mol of radioactive cyanate per peptide chain concomitant with inactivation. Analysis of acid hydrolysates of the radioactive enzyme reveals that lysyl residues are the sole amino acids modified. These results suggest that cyanate, or isocyanic acid, may bind to the active site of this enzyme as an analogue of carbon dioxide and carbamylate a lysyl residue at the active site.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D003485:Cyanates / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D007523:Isocitrates / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine; D008433:Mathematics; D009206:Myocardium / Q000201:enzymology*; D009243:NAD; D010084:Oxidation-Reduction; D011485:Protein Binding; D013552:Swine; D013997:Time Factors
D003485:Cyanates; D007523:Isocitrates; D009243:NAD; D000244:Adenosine Diphosphate; D004492:Edetic Acid; D007521:Isocitrate Dehydrogenase; D008239:Lysine