Kinetic and physiological effects of alterations in homologous isocitrate-binding sites of yeast NAD(+)-specific isocitrate dehydrogenase

Yeast NAD(+)-specific isocitrate dehydrogenase is an allosterically regulat ed octameric enzyme composed of four each of two homologous but nonidentica l subunits designated IDH1 and IDH2. Models based on the crystallographic s tructure of Escherichia coli isocitrate dehydrogenase suggest that both yea st subunits contain isocitrate-binding sites. Identities in nine residue po sitions are predicted for the IDH2 site whereas four of the nine positions differ between the IDH1 and bacterial enzyme sites. Thus, we speculate that the IDH2 site is catalytic and that the IDH1 site may bind but not catalyt ically alter isocitrate. This was examined by kinetic analyses of enzymes w ith independent and concerted replacement of residues in each yeast IDH sub unit site with the residues that differ in the other subunit site. Mutant e nzymes were expressed in a yeast strain containing disrupted IDH1 and IDH2 loci and affinity-purified for kinetic analyses. The primary effects of var ious residue replacements in IDH2 were reductions of 30-> 300-fold in V-max values, consistent with the catalytic function of this subunit. In contras t, replacement of all four residues in IDH1 produced a 17-fold reduction in V-max under the same assay conditions, suggesting that the IDH1 site is no t the primary catalytic site. However, single or multiple residue replaceme nts in IDH1 uniformly increased half-saturation concentrations for isocitra te, implying that isocitrate can be bound at this site. Both subunits appea r to contribute to cooperativity with respect to isocitrate, but AMP activa tion is lost only with residue replacements in IDH1. Overall, results are c onsistent with isocitrate binding by IDH2 for catalysis and with isocitrate binding by IDH1 being a prerequisite for allosteric activation by AMP. The effects of residue substitutions on enzyme function in vivo were assessed by analysis of various growth phenotypes. Results indicate a positive corre lation between the level of IDH catalytic activity and the ability of cells to grow with acetate or glycerol as carbon sources. In addition, lower lev els of activity are associated with increased production of respiratory-def icient (petite) segregants.