Study of the functions of the 3 different subunits of human NAD-isocitrate dehydrogenase

This dissertation describes three studies on the human NAD-dependent isocitrate dehydrogenase (IDH), a heterotetrameric mitochondrial enzyme with 2alpha:1beta:1gamma subunit ratio. The three subunits share 40-52% identity in amino acid sequence and each includes a tyrosine in a comparable position: alphaY126, betaY137 and gammaY135. In the first study to examine the role of the corresponding tyrosines of each of the subunits of human NAD-IDH, the tyrosines were mutated (one subunit at a time) to Ser, Phe or Glu. Enzymes were expressed with one mutant and two wild-type subunits. The results of characterization of the mutant enzymes suggest that betaY137 is involved in NAD binding and allosteric activation by ADP. The alphaY126 is required for catalytic activity and likely acts as a general acid in the reaction. The gammaY135 is also required for catalytic activity and may be involved in proper folding of the enzyme. The corresponding tyrosines in the three dissimilar subunits of NAD-IDH thus have distinctive functions.;In the second study, we describe two families with retinitis pigmentosa, a hereditary neurodegeneration of rod and cone photoreceptors in the retina. Affected family members were homozygous for loss-of-function mutations in IDH3B, encoding the beta-subunit of NAD-specific isocitrate dehydrogenase (NAD-IDH, or IDH3),which is believed to catalyze the oxidation of isocitrate to alpha-ketoglutarate in the citric acid cycle. Cells from affected individuals had a substantial reduction of NAD-IDH activity, with about a 300-fold increase in the Km for NAD. NADP-specific isocitrate dehydrogenase (NADP-IDH, or IDH2), an enzyme that catalyzes the same reaction, was normal in affected individuals, and they had no health problems associated with the enzyme deficiency except for retinitis pigmentosa. These findings support the hypothesis that mitochondrial NADP-IDH, rather than NAD-IDH, serves as the main catalyst for this reaction in the citric acid cycle outside the retina, and that the retina has a particular requirement for NAD-IDH.;In the third study, we separately expressed in bacteria and purified the alpha and gamma subunits of NAD-IDH. We studied the characteristics of the alphagamma complex and compared them to the properties of the complete wild-type enzyme as well as a previously studied complete mutant enzyme of gamma subunit (gammaR97Q) which had wild-type alpha and beta subunits. Our results indicate that the alpha and gamma subunits alone are inactive. The kinetic properties of the wild-type alphagamma complex indicate that these two subunits are sufficient for efficient binding isocitrate, Mn 2+, NAD and ADP, but the Vmax suggests that all three types of subunits are required for maximum activity. The gammaR97Q whole enzyme mutant had a high Km for NAD and a loss of allosteric activation due to ADP. Since the wild-type alphagamma complex exhibited Km values comparable to those of the wild-type, it is possible that the mutant gammaR97Q subunit interacts improperly with the other two wild-type subunits. The mutant complex of alphagammaR97Q was entirely inactive which confirms the possibility of improper interaction of mutated gamma with the other two subunits. It also proves that the activity of the whole enzyme mutant aR97Q can be attributed to the wild-type alpha and beta subunits which are present.