The Role of Sulfhydryl Groups in the Catalytic Function of Isocitrate Dehydrogenase: III. EFFECT OF N-ETHYLMALEIMIDE ON CHEMICAL AND PHYSICAL PROPERTIES

Abstract

Abstract Incubation of the TPN-dependent isocitrate dehydrogenase from pig heart with 1-14C-N-ethylmaleimide (NEM) leads to inactivation and altered Michaelis constants for isocitrate and oxalosuccinate concomitant with incorporation of 2 moles of reagent. Reaction of enzyme with radioactive NEM in the presence of isocitrate and Mn++ yields a catalytically functional enzyme, with the same altered Michaelis constants, which also contains 2 moles of reagent. Inactive enzyme with a single carboxymethyl methionyl residue exhibits the same ability to bind radioactive NEM as does native enzyme; however, prior inactivation by reaction of 5 sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) prevents binding of NEM. Cysteine is the sole type of amino acid modified in the inactive NEM-enzyme, as determined by paper chromatographic identification of radioactive cysteinosuccinic acid and S-(N-ethylsuccinimido)cysteine in acid and proteolytic digests of 14C-labeled modified enzyme. Thirteen moles of cysteic acid are found after performic acid oxidation of native enzyme, the same as the number of free —SH groups observed by reaction with DTNB after denaturation; therefore, no disulfide bonds are present in isocitrate dehydrogenase. A decrease in the number of measurable —SH groups after reaction with NEM is consistent with modification of cysteinyl residues. By contrast, the recovery of NH2-terminal alanine after the Edman reaction was approximately the same for native and modified enzyme. Inactive enzymes with a single carboxymethyl methionyl residue, with 2 or 5 altered sulfhydryl groups, give a reaction of antigenic identity to that of native enzyme as measured against rabbit antibody to isocitrate dehydrogenase. The molecular size of the inactive NEM-enzyme is the same as that of native enzyme, as determined by gel filtration on Sephadex G-150, and the rates of proteolytic digestion by Pronase are not significantly different. These data argue against a generalized structural change in the modified enzyme. However, a small conformational change is indicated by a decrease in the reaction rate of DTNB with the residual —SH groups of the inactive NEM-enzyme. The magnitude of conformational change is not directly related to loss in enzymatic activity, since an observed decrease in the amplitude of the optical rotatory dispersion curves is greater for active NEM-enzyme ([α]234 -5610°) than it is for inactive NEM-enzyme ([α]234 -7430°), as compared to native isocitrate dehydrogenase ([α]234 -8050°). These results, in conjunction with those of the preceding paper, suggest that, in the absence of isocitrate and MnSO4, modification of Sulfhydryl Group A produces inactivation, whereas modification of Sulfhydryl Group B leads to a small conformational change which is reflected in altered Michaelis constants. In the presence of substrates, NEM reacts with 2 sulfhydryl groups, including Group B but not Group A, which produces a further change in the conformation of the enzyme but no concomitant change in the kinetic properties.