Abstract
Understanding the factors that determine the catalytic efficiency of cellulases is of considerable importance in cellulosic ethanol production, especially at high temperature. The cellulase 12A from the hyperthermophile Thermotoga maritima (TmCel12A) is a possible candidate for accelerating the rate of hydrolysis via temperature elevation up to as high as 95 °C. However, the details of the catalytic mechanism and origin of the activity of TmCel12A at high temperature have not been well studied. Here, the enzyme-catalyzed reaction is explored using free energy simulations (potential of mean force) with umbrella sampling and quantum mechanical/molecular mechanical (SCC-DFTB/MM) potential at both relatively low (37 °C) and high (85 °C) temperatures. The free energy barriers for glycosylation and deglycosylation are calculated to be 22.5 ± 0.4 and 24.5 ± 0.7 kcal · mol−1 at 85 °C, respectively. The barrier for deglycosylation is found to decrease with increasing temperature or as a result of the Y61 → G mutation, consistent with experimental observations. The transition state for glycosylation and deglycosylation obtained from the simulations is in an oxocarbonium state with the −1 glucose ring having an E3 envelop (or 4H3 half-chair) conformation. A unique characteristic of the TmCel12A structure seems to be the existence of a stable moiety that may play a role in “holding” cellulose at the binding site with the correct orientation for the reaction even at 85 °C. This stable moiety (comprising hydrogen-bonded E116, E134, E227 and an active-site water molecule) may be one of the important factors for the relatively high activity of TmCel12A at high temperature.
