Mechanism for Activation of Triosephosphate Isomerase by Phosphite Dianion: The Role of a Ligand-Driven Conformational Change M. Merced Malabanan Tina L. Amyes John P. Richard 10.1021/ja208019p.s001 https://acs.figshare.com/articles/journal_contribution/Mechanism_for_Activation_of_Triosephosphate_Isomerase_by_Phosphite_Dianion_The_Role_of_a_Ligand_Driven_Conformational_Change/2599867 The L232A mutation in triosephosphate isomerase (TIM) from Trypanosoma brucei brucei results in a small 6-fold decrease in <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> for the reversible enzyme-catalyzed isomerization of glyceraldehyde 3-phosphate to give dihydroxyacetone phosphate. In contrast, this mutation leads to a 17-fold <i>increase</i> in the second-order rate constant for the TIM-catalyzed proton transfer reaction of the truncated substrate piece [1-<sup>13</sup>C]glycolaldehyde ([1-<sup>13</sup>C]-GA) in D<sub>2</sub>O, a 25-fold <i>increase</i> in the third-order rate constant for the reaction of the substrate pieces GA and phosphite dianion (HPO<sub>3</sub><sup>2–</sup>), and a 16-fold <i>decrease</i> in <i>K</i><sub>d</sub> for binding of HPO<sub>3</sub><sup>2–</sup> to the free enzyme. Most significantly, the mutation also results in an 11-fold <i>decrease</i> in the extent of activation of the enzyme toward turnover of GA by bound HPO<sub>3</sub><sup>2–</sup>. 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<sub>c</sub>) relative to an inactive open form (E<sub>o</sub>). 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<sub>c</sub> relative to E<sub>o</sub>. 2011-10-19 00:00:00 TIM side chain D 2O enzyme triosephosphate isomerase phosphite dianion substrate pieces GA L 232A mutation Eo L 232A Ec Trypanosoma brucei brucei results Phosphite Dianion Triosephosphate Isomerase carboxylate side chain dihydroxyacetone phosphate HPO steric interactions