The K12G mutation at yeast triosephosphate isomerase (TIM) results in a 5.5 x 10(5)-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 x 10(6) 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.