Mechanism for Activation of Triosephosphate Isomerase by Phosphite Dianion: The Role of a Ligand-Driven Conformational Change

The L232A mutation in triosephosphate isomerase (TIM) from Trypanosoma brucei brucei results in a small 6-fold decrease in k_cat/K_m for the reversible enzyme-catalyzed isomerization of glyceraldehyde 3-phosphate to give dihydroxyacetone phosphate. In contrast, this mutation leads to a 17-fold increase in the second-order rate constant for the TIM-catalyzed proton transfer reaction of the truncated substrate piece [1-¹³C]glycolaldehyde ([1-¹³C]-GA) in D₂O, a 25-fold increase in the third-order rate constant for the reaction of the substrate pieces GA and phosphite dianion (HPO₃^2–), and a 16-fold decrease in K_d for binding of HPO₃^2– to the free enzyme. Most significantly, the mutation also results in an 11-fold decrease in the extent of activation of the enzyme toward turnover of GA by bound HPO₃^2–. The data provide striking evidence that the L232A mutation leads to a ca. 1.7 kcal/mol stabilization of a catalytically active loop-closed form of TIM (E_c) relative to an inactive open form (E_o). We propose that this is due to the relief, in L232A mutant TIM, of unfavorable steric interactions between the bulky hydrophobic side chain of Leu-232 and the basic carboxylate side chain of Glu-167, the catalytic base, which destabilize E_c relative to E_o.