Location of the Coenzyme Binding Site in the Porcine Mitochondrial NADP-dependent Isocitrate Dehydrogenase

J Biol Chem. 2005 Aug 26;280(34):30349-53. doi: 10.1074/jbc.M505828200. Epub 2005 Jun 23.


The structure of crystalline porcine mitochondrial NADP-dependent isocitrate dehydrogenase (IDH) has been determined in complex with Mn2+-isocitrate. Based on structural alignment between this porcine enzyme and seven determined crystal structures of complexes of NADP with bacterial IDHs, Arg83, Thr311, and Asn328 were chosen as targets for site-directed mutagenesis of porcine IDH. The circular dichroism spectra of purified wild-type and mutant enzymes are similar. The mutant enzymes exhibit little change in Km for isocitrate or Mn2+, showing that these residues are not involved in substrate binding. In contrast, the Arg83 mutants, Asn328 mutants, and T311A exhibit 3-20-fold increase in the Km(NADP). We propose that Arg83 enhances NADP affinity by hydrogen bonding with the 3'-OH of the nicotinamide ribose, whereas Asn328 hydrogen bonds with N1 of adenine. The pH dependence of Vmax for Arg83 and Asn328 mutants is similar to that of wild-type enzyme, but for all the Thr311 mutants, pK(es) is increased from 5.2 in the wild type to approximately 6.0. We have previously attributed the pH dependence of Vmax to the deprotonation of the metal-bound hydroxyl of isocitrate in the enzyme-substrate complex, prior to the transfer of a hydride from isocitrate to NADP's nicotinamide moiety. Thr311 interacts with the nicotinamide ribose and is the closest of the target amino acids to the nicotinamide ring. Distortion of the nicotinamide by Thr311 mutation will likely be transmitted to Mn2+-isocitrate resulting in an altered pK(es). Because porcine and human mitochondrial NADP-IDH have 95% sequence identity, these results should be applicable to the human enzyme.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenine / chemistry
  • Amino Acid Sequence
  • Animals
  • Arginine / chemistry
  • Asparagine / chemistry
  • Binding Sites
  • Circular Dichroism
  • Codon
  • Crystallography, X-Ray
  • Cytosol / metabolism
  • DNA, Complementary / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Hydrogen-Ion Concentration
  • Isocitrate Dehydrogenase / chemistry*
  • Isocitrates / chemistry
  • Kinetics
  • Magnesium / chemistry
  • Mitochondria / enzymology*
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation
  • NADP / chemistry
  • Niacinamide / chemistry
  • Oligonucleotides / chemistry
  • Protein Binding
  • Swine
  • Threonine / chemistry


  • Codon
  • DNA, Complementary
  • Isocitrates
  • Oligonucleotides
  • Niacinamide
  • Threonine
  • NADP
  • Asparagine
  • Arginine
  • isocitric acid
  • Isocitrate Dehydrogenase
  • isocitrate dehydrogenase (NADP+)
  • Magnesium
  • Adenine