Role of Ligand-Driven Conformational Changes in Enzyme Catalysis: Modeling the Reactivity of the Catalytic Cage of Triosephosphate Isomerase

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, 28683550J. Am. Chem. Soc.20171391051410525). 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.

Protein Flexibility and Stiffness Enable Efficient Enzymatic Catalysis

“…This prediction from experiments was confirmed by the results of empirical valence bond (EVB) calculations, 14 which give similar activation barriers ( Scheme 8 ) for the TIM-catalyzed deprotonation of GAP [(Δ G ⧧ ) GAP = 12.9 ± 0.8 kcal/mol], for deprotonation of the substrate piece GA [(Δ G ⧧ ) GA = 15.0 ± 2.4 kcal/mol], and for deprotonation of the pieces GA·HP i [(Δ G ⧧ ) GA·HPi = 15.5 ± 3.5 kcal/mol]. 14 We concluded that the closed form of TIM created by protein–dianion binding interactions is competent to carry out fast deprotonation of the carbon acid whole substrate or the substrate piece GA. The effect of the enzyme-bound dianion on Δ G ⧧ for reaction of the active closed enzyme is small (≤2.6 kcal/mol), in comparison to the larger 12 and 5.8 kcal/mol intrinsic phosphodianion and phosphite dianion-binding energy that is utilized in stabilization of the transition states for TIM-catalyzed deprotonation of GAP and GA·HP i , respectively.…”

“…Many results are consistent with the conclusion that the structures for reactive Michaelis complexes of enzyme catalysts are stiff and allow for minimal protein motions away from highly organized forms. As noted above, enzyme-ligand complexes from X-ray crystallographic analyses serve as good starting points for calculations that model the experimental activation barrier for turnover at enzyme active sites, 14 , 15 so that the stiffness of reactive enzyme–substrate complexes is similar to that for crystalline enzymes. The empirical valence bond (EVB) computational methods developed by Arieh Warshel strongly emphasize the modeling of electrostatic interactions.…”

“…These structures are routinely used in high-level calculations of activation barriers for formation of enzyme-bound transition states that are in good agreement with the experimental activation barriers. 14 − 19 This suggests that the rigid structures capture the full catalytic power of many enzymes.…”

Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-Phosphate Dehydrogenase

“…Following from our previous recent studies of the proton transfer reactions catalyzed by triosephosphate isomerase, in the present work, we used the empirical valence bond (EVB) approach 26 to model the chemical reaction catalyzed by hlGPDH (Figure 1), using a similar protocol as that described in our previous work. [28][29][30][31] Specifically, we modeled the hydride transfer reaction based on the valence bond states shown in Figure 2. Simulations were performed of the reaction catalyzed by wild-type hlGPDH, as well as by the K120A, Q295A, and R269A variants of this enzyme (i.e.…”

“…33 The system was then described using a standard multi-layer approach in which all residues within the inner 85% of the water sphere were fully mobile, those in the outer 15% were subjected to a 10 kcal mol -1 Å -2 harmonic positional restraint to restrain them to their original crystallographic positions, and all residues outside the droplet were essentially fixed to their crystallographic positions using a 200 kcal mol -1 Å -2 restraint, as in our previous work. [28][29][30][31] All residues within the mobile region (i.e. the inner 85% of the water sphere) were assigned their expected ionization states at physiological pH, based on a combination of empirical screening using PROPKA 3.1 34 and visual inspection.…”