AFFINITY PURIFICATION AND KINETIC-ANALYSIS OF MUTANT FORMS OF YEAST NAD(-SPECIFIC ISOCITRATE DEHYDROGENASE())

Polyhistidine tags were added to the carboxyl termini of the two homol ogous subunits of yeast NAD(+)-specific isocitrate dehydrogenase (IDH) . The tag in either the IDH1 or IDH2 subunit permits one step affinity purification from yeast cellular extracts of catalytically active and allosterically responsive holoenzyme. This expression system was used to investigate subunit-specific contributions of residues with putati ve functions in adenine nucleotide binding, The primary effect of simu ltaneous replacement of the adjacent Asp-279 and Ile-280 residues in I DH1 with alanines is a dramatic loss of activation by AMP. In contrast , alanine replacement of the homologous Asp-286 and Ile-287 residues i n IDH2 does not alter the allosteric response to AMP, but produces a 1 60-fold reduction in V-max due to a 70-fold increase in the S-0.5 valu e for NAD(+), These results suggest that the targeted aspartate/isoleu cine residues may con tribute to regulator binding in IDH1 and to cofa ctor binding in IDHS, i.e. that these homologous residues are located in regions that have evolved for binding the adenine nucleotide compon ents of different ligands, In other mutant enzymes, an alanine replace ment of Asp 191 in IDH1 eliminates measurable catalytic activity, and a similar substitution of the homologous Asp-197 in IDH2 produces plei otropic catalytic effects, A model is presented for the primary functi on of IDH2 in catalysis and of IDH1 in regulation, with crucial roles for these single aspartate residues in the communication and functiona l interdependence of the two subunits.