Abstract
We have previously performed empirical valence bond calculations of the kinetic activation barriers, ΔG‡calc, for the deprotonation of complexes between TIM and the whole substrate glyceraldehyde-3-phosphate (GAP, Kulkarni et al.J. Am. Chem. Soc.2017, 139, 10514–1052528683550). We now extend this work to also study the deprotonation of the substrate pieces glycolaldehyde (GA) and GA·HPi [HPi = phosphite dianion]. Our combined calculations provide activation barriers, ΔG‡calc, for the TIM-catalyzed deprotonation of GAP (12.9 ± 0.8 kcal·mol–1), of the substrate piece GA (15.0 ± 2.4 kcal·mol–1), and of the pieces GA·HPi (15.5 ± 3.5 kcal·mol–1). The effect of bound dianion on ΔG‡calc is small (≤2.6 kcal·mol–1), in comparison to the much larger 12.0 and 5.8 kcal·mol–1 intrinsic phosphodianion and phosphite dianion binding energy utilized to stabilize the transition states for TIM-catalyzed deprotonation of GAP and GA·HPi, respectively. This shows that the dianion binding energy is essentially fully expressed at our protein model for the Michaelis complex, where it is utilized to drive an activating change in enzyme conformation. The results represent an example of the synergistic use of results from experiments and calculations to advance our understanding of enzymatic reaction mechanisms.
Highlights
Triosephosphate isomerase (TIM) catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to (R)-glyceraldehyde 3-phosphate (GAP), through enzyme-bound cis-enediolate reaction intermediates (Scheme 1A),[4] and phosphite dianion-activated deprotonation of Scheme 1. (A) Mechanism for the TIM-Catalyzed Reaction; (B) TIM-Catalyzed Reactions of GAP and the Substrate Pieces GA·HPi
Model for the TIM-Catalyzed Reaction of the Substrate Pieces that Separates the Enzyme Conformational Change (Kc) from Deprotonation of Bound Substrate concentrations in the absence of the activating dianion (Kc ≪ 1): the dianion binding interactions are utilized to mold the inactive open enzyme (EO) into the structured and fully active catalyst,1a,b as described originally in Koshland’s induced fit model.[7]. Once these binding interactions are expressed at EC, the dianion becomes a spectator that does not affect the reactivity of the truncated carbon acid GA [(kcat/Km)E′ =EHPi, Scheme 2]
The energetic price to formation of EC is paid for by utilization of binding interactions with nonreacting substrate fragments.1a. These binding interactions are assumed to be fully expressed at ΔECG, ‡a,nfodr cannot act to reduce the TIM-catalyzed proton transfer from activation barrier, the carbon acid to the carboxylate side chain of E165
Summary
This focus on the Michaelis complex neglects to consider the mechanism for the utilization of the large intrinsic binding energy of nonreacting substrate fragments in transition state stabilization,[3] which gives rise to dianion activation of the reaction of the truncated substrate piece.1a The present computational studies strongly suggest that the total intrinsic dianion binding energy is essentially entirely expressed at the Michaelis complex to the substrate pieces (Table 1) This provides strong support for the conclusion that the difference between the intrinsic and the observed dianion binding energy is equal to the binding energy utilized to drive an uphill enzyme conformational change during formation of the Michaelis complex (Scheme 2).[1] We are working on developing computational protocols to formally calculate the dianion binding energy that is utilized to drive the conformational change of TIM.
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