BPS2026 – Activation heat capacity and dynamical networks in thermoadaptation: Triosephosphate isomerase

Heat capacity changes are increasingly recognized as crucial factors for understanding the temperature dependence of enzyme-catalyzed reaction rates. We combine simulations and experiment to investigate triosephosphate isomerase (TIM, TPI) from psychrophilic (cold-TIM), mesophilic (meso-TIM), and thermophilic (hot-TIM) organisms. Kinetic data show clear curvature in rate-temperature plots, particularly for the psychrophilic enzyme, independent of unfolding. This non-Arrhenius behavior is accounted for by macromolecular rate theory (MMRT), implying an activation heat capacity, largest for cold-TIM. Extensive molecular dynamics (MD) simulations reveal the dynamical origins of these activation heat capacity differences, with calculated values in the same order as experiment, i.e., the largest (most negative) for cold-TIM and the smallest for hot-TIM. Significant changes in conformational populations are found between reactant and transition state analog complex in both cold- and meso-TIM. The results identify a transition-state-like conformation (TLC), with important differences between the Michaelis complex and transition state-like complex in dynamics across the whole dimeric enzyme, including solvent-exposed surfaces, dimer interfaces, and loop regions and helices near the active sites. In cold-TIM, striking differences in correlated dynamics, spanning the entire enzyme dimer, are observed between these states; meso-TIM shows smaller changes in correlated dynamics, and hot-TIM shows very little difference, consistent with the predictions of MMRT. Dynamical non-equilibrium molecular dynamics (D-NEMD) simulations and shortest path map (SPM) analysis identify specific communication networks and individual residues throughout the protein that enable efficient catalysis at different temperatures. These identified regions and residues provide targets for protein engineering to rationally shift enzyme temperature optima. Dynamical differences between crucial states along the reaction pathway play a key role in determining the optimum temperature of activity for this archetypal enzyme.