Rescue of K12G Triosephosphate Isomerase by Ammonium Cations: The Reaction of an Enzyme in Pieces

Abstract

The K12G mutation at yeast triosephosphate isomerase (TIM) results in a 5.5 × 10⁵-fold decrease in k_cat/K_m for isomerization of glyceraldehyde 3-phosphate, and the activity of this mutant can be successfully “rescued” by NH₄⁺ and primary alkylammonium cations. The transition state for the K12G mutant TIM-catalyzed reaction is stabilized by 1.5 kcal/mol by interaction with NH₄⁺. The larger 3.9 kcal/mol stabilization by CH₃CH₂CH₂CH₂NH₃⁺ is due to hydrophobic interactions between the mutant enzyme and the butyl side chain of the cation activator. There is no significant transfer of a proton from alkylammonium cations to GAP at the transition state for the K12G mutant TIM-catalyzed reaction, because activation by a series of RNH₃⁺ shows little or no dependence on the pK_a of RNH₃⁺. A comparison of k_cat/K_m = 6.6 × 10⁶ M⁻¹ s⁻¹ for the wildtype TIM-catalyzed isomerization of GAP and the third-order rate constant of 150 M⁻² s⁻¹ for activation by NH₄⁺ of the K12G mutant TIM-catalyzed isomerization shows that stabilization of the bound transition state by the effectively intramolecular interaction of the cationic side chain of Lys-12 at wildtype TIM is 6.3 kcal/mol greater than that for the corresponding intermolecular interaction of NH₄⁺ at K12G mutant TIM.