Effect of Modification of a Methionyl Residue on the Kinetic and Molecular Properties of Isocitrate Dehydrogenase

Abstract Pig heart isocitrate dehydrogenase, which catalyzes the TPN-dependent dehydrogenation of isocitrate as well as the decarboxylation of oxalosuccinate, is inactivated by treatment with iodoacetate at pH 5.5 and 30°. Approximately 1 mole of 14C-labeled reagent is incorporated per mole of protein. The effect of alkylation on the isocitrate dehydrogenase activity is greater than that on the oxalosuccinate decarboxylase activity of the enzyme, and therefore the active sites for these two catalytic functions are distinguishable. Inactivation can be prevented by the inclusion of isocitrate in the reaction mixture, suggesting that iodoacetate causes a specific modification in the functional region of the enzyme. Vmax for both the native and the partially modified enzyme depends on the unprotonated form of an ionizable group of pK 5.4, which shifts to pK 6.3 when measured in 20% ethanol as solvent. A requirement for the dissociable form of a carboxyl group is suggested. The Michaelis constants of partially alkylated enzymes are unchanged from those of the native enzyme. It is concluded that iodoacetate alters an amino acid residue essential for the dehydrogenase reaction. Iodoacetate treatment does not produce marked structural alterations in the enzyme, since no differences in molecular weight or in the ultraviolet absorption or optical rotatory dispersion spectra were detected. The pseudo-first order rate constant for inactivation of the enzyme by iodoacetate decreases from pH 7.8 to 7.2, is almost constant from pH 7.2 to 6.1, and increases markedly below pH 6.0. Enzyme alkylated at pH 5.6 exhibits the same sulfhydryl content as the native enzyme, and an unaltered histidine, lysine, and tyrosine composition. Amino acid analysis indicates that the methionine content of the alkylated enzyme is decreased by 1 residue as compared to the native enzyme, and a new peak, that of carboxymethylhomocysteine, is observed. Paper chromatography of proteolytic digests of 14C-labeled alkylated enzyme confirms the conclusion that alkylation of 1 methionine residue is responsible for the altered activity of isocitrate dehydrogenase. The rate of inactivation at pH 5.6 is independent of ionic strength. Modified enzyme exhibits a mobility identical with that of native enzyme in cellulose acetate electrophoresis from pH 6.2 to 9.5. These observations are consistent with attack of iodoacetate on an uncharged methionyl residue to yield dipolar carboxymethylmethionine sulfonium salt.