Understanding the origin of changes in activity of a cellulase from Thermotoga maritima in binary imidazolium acetate/water mixtures.

The family 5 glycoside hydrolase from the hyperthermophilic bacteria Thermotoga maritima (Tm_Cel5A) is a thermally stable enzyme with an optimal temperature of 70 °C and a melting temperature in excess of 90 °C. The activity of Tm_Cel5A decreases in binary mixtures of water and the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C₂C₁Im][OAc]), which has been shown to be very effective at pretreating biomass as the [C₂C₁Im][OAc] concentration increases. Despite the existence of the X-ray structure of Tm_Cel5A and detailed biochemical characterization in binary H₂O:[C₂C₁Im][OAc] solutions, no theoretical description of the reaction mechanism with complete representation of the enzyme/substrate and the necessary exhaustive sampling of the relevant phase space has been reported. As a result, no rigorous atomic-level explanation for the observed gradual loss of activity with increasing concentration of [C₂C₁Im][OAc] has been given.

    The study to be discussed couples quantum chemical investigations of the potential energy surface of the enzymatic reaction in both solution and active-site cluster models, with detailed molecular mechanics simulations of the reacting enzyme-substrate system using the empirical valence bond and free energy perturbation methods. These calculations allow elucidation of the chemical origins of the catalytic power of the enzyme and provide atomistic insight into the effect of the ionic liquid. The resulting understanding can be leveraged to suggest beneficial mutations for tuning the activity of the enzyme, which can be directly tested by experiment and may lead to improved enzyme mixtures for degradation of polysaccharides in the presence of higher quantities of [C₂C₁Im][OAc].