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

摘要

The K12G mutation at yeast triosephosphate isomerase (TIM) results in a 5.5 × 10⁵-fold decrease in kcat/Km 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 pKa of RNH3⁺. A comparison of kcat/Km = 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 for the corresponding intermolecular interaction of NH4⁺ at K12G mutant TIM.