Evolution of a transition state: Role of lys100 in the active site of isocitrate dehydrogenase

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. Same but different: In isocitrate dehydrogenase, the substrate C4-carboxylate abuts the positively charged nicotin- amide of the coenzyme. Lys100 stabilizes the oxyanion formed on the nicotin- amide 3-carboxamide so that hydride transfer can occur without neutralizing the positive charge on the nicotinamide. This transition state differs in related dehydrogenases without this lysine.