Reaction of essential lysyl residues of pig heart diphosphopyridine nucleotide dependent isocitrate dehydrogenase with 2,4-pentanedione

The rate of inactivation of pig heart DPN-specific isocitrate dehydrogenase by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration. Isocitrate incombination with manganous ion can prevent inactivation, and a dissociation constant (KIC) for the enzyme-isocitrate complex can be calculated which is similar in magnitude to the Km for isocitrate under the same conditions. Although neither the cofactor,DPN, nor the allosteric activator, ADP, prevents inactivation by reagent, ADP lowers both KIC and Km to the same extent. These data suggest that the reagent may be reacting with residues within a binding site for manganeous-isocitrate. DPNH accelerates the inactivation and also enhances protection by isocitrate, lowering KIC by a factor of 20. Because ADP does not prevent the DPNH rate enhancement, it is unlikely that the two nucleotides compete for identical binding sites. Reaction with 2,4-pentanedione thus provides a probe of the mode of ligand interaction with the enzyme. Inactivation appears to result from the reaction of 2,4-pentanedione with lysyl residues to form enamines. The occurrence of a new absorbance band during inactivation and the isolation by gel filtration of enzyme with an absorbance peak at 312 nm are consistent with enamine formation. Hydroxylamine, which abolishes the 312-nm peak, also causes appreciable reactivation of the enzyme. By use of [2,4-14C]-2,4-pentanedione, it was established that reaction of an average of no more than 3 lysines of the 26 per peptide chain resulted in complete inactivation; and an average of only 2 lysines react when enzymatic activity is retained in the presence of 50 mM isocitrate. Reaction with arginine was excluded by the unchanged amino acid composition of modified enzyme. These data suggest that formation of an enamine of possibly 1, and certainly no more than 3, lysine residue(s) in the catalytic center of the enzyme is responsible for inactivation by 2,4-pentanedione.