Get access

Evolution of a Transition State: Role of Lys100 in the Active Site of Isocitrate Dehydrogenase

Authors

  • Dr. Stephen P. Miller,

    1. Biotechnology Institute, The University of Minnesota, 1479 Gortner Avenue, St. Paul, MN 55108 (USA)
    Search for more papers by this author
  • Dr. Susana Gonçalves,

    1. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127, 2780 Oeiras (Portugal)
    2. Currently at The European Synchotron Radiation Facility, 6 Rue Jules Horowitz, B. P. 220, 38043 Grenoble CEDEX 9 (France)
    Search for more papers by this author
  • Prof. Pedro M. Matias,

    1. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127, 2780 Oeiras (Portugal)
    2. Instituto de Biologica Experimental e Tecnológia, Av. da República, EAN, 2780 Oeiras (Portugal)
    Search for more papers by this author
  • Prof. Antony M. Dean

    Corresponding author
    1. Biotechnology Institute, The University of Minnesota, 1479 Gortner Avenue, St. Paul, MN 55108 (USA)
    2. Department of Ecology, Evolution and Behavior, The University of Minnesota, 1987 Buford Circle, St. Paul, MN 55108 (USA)
    • Biotechnology Institute, The University of Minnesota, 1479 Gortner Avenue, St. Paul, MN 55108 (USA)===

    Search for more papers by this author

Abstract

An active site lysine essential to catalysis in isocitrate dehydrogenase (IDH) is absent from related enzymes. As all family members catalyze the same oxidative β-decarboxylation at the (2R)-malate core common to their substrates, it seems odd that an amino acid essential to one is not found in all. Ordinarily, hydride transfer to a nicotinamide C4 neutralizes the positive charge at N1 directly. In IDH, the negatively charged C4-carboxylate of isocitrate stabilizes the ground state positive charge on the adjacent nicotinamide N1, opposing hydride transfer. The critical lysine is poised to stabilize—and perhaps even protonate—an oxyanion formed on the nicotinamide 3-carboxamide, thereby enabling the hydride to be transferred while the positive charge at N1 is maintained. IDH might catalyze the same overall reaction as other family members, but dehydrogenation proceeds through a distinct, though related, transition state. Partial activation of lysine mutants by K+ and NH4+ represents a throwback to the primordial state of the first promiscuous substrate family member.

Ancillary