Enzyme surface rigidity tunes the temperature dependence of catalytic rates.

The structural origin of enzyme adaptation to low temperature, allowing efficient catalysis of chemical reactions even near the freezing point of water, remains a fundamental puzzle in biocatalysis. A remarkable universal fingerprint shared by all cold-active enzymes is a reduction of the activation enthalpy accompanied by a more negative entropy, which alleviates the exponential decrease in chemical reaction rates caused by lowering of the temperature. Herein, we explore the role of protein surface mobility in determining this enthalpy-entropy balance. The effects of modifying surface rigidity in cold- and warm-active trypsins are demonstrated here by calculation of high-precision Arrhenius plots and thermodynamic activation parameters for the peptide hydrolysis reaction, using extensive computer simulations. The protein surface flexibility is systematically varied by applying positional restraints, causing the remarkable effect of turning the cold-active trypsin into a variant with mesophilic characteristics without changing the amino acid sequence. Furthermore, we show that just restraining a key surface loop causes the same effect as a point mutation in that loop between the cold- and warm-active trypsin. Importantly, changes in the activation enthalpy-entropy balance of up to 10 kcal/mol are almost perfectly balanced at room temperature, whereas they yield significantly higher rates at low temperatures for the cold-adapted enzyme.