THE ROLE OF DISSIMILAR SUBUNITS OF NAD-SPECIFIC ISOCITRATE DEHYDROGENASE FROM PIG-HEART - EVALUATION USING AFFINITY LABELING

NAD-specific isocitrate dehydrogenase from pig heart is composed of 3 dissimilar subunits present in the native enzyme as 2.alpha.:1.beta.:1.gamma., with a tetramer being the smallest form of complete enzyme. The role of these subunits was explored using affinity labeling. Specifically labeled subunits are separated and then recombined with unmodified subunits to form dimers. Recombination of .beta. or .gamma. subunits modified by the isocitrate analogs, 3-bromo-2-ketoglutarate and 3,4-didehydro-2-ketoglutarate, with unmodified .alpha. subunit led to the same activity in the dimer as when unmodified .beta. or .gamma. was combined with .alpha.. Contrastingly, modification of .alpha. with these isocitrate analogs led to loss in activity either alone or when recombined with .beta. or .gamma.. Hence, the isocitrate site on .alpha. is required for catalytic activity but the isocitrate sites on .beta. or .gamma. are not necessary for the activity of the functional dimer. Reaction of isolated subunits with 3-bromo-2-ketoglutarate shows that .alpha. and the .alpha..beta. dimer are modified .apprx. the same rate as holoenzyme, suggestive of similarity of the isocitrate site in native enzyme and in isolated active entities containing .alpha. subunit; in contrast, .beta. and .gamma. subunits react more slowly. Modification by the 2'',3''-dialdehyde derivative of the allosteric effector, ADP, led to loss of activity in reconstituted dimers, independent of which subunit was modified. Reaction of isolated subunits with the dialdehyde derivative of ADP is slow compared to the initial reaction with native enzyme, indicating differences in the effects of ADP on intact enzyme and subunits. The ADP sites on all subunits may thus be important in intersubunit interactions, which in turn modulate catalytic activity.