Enzyme surface rigidity tunes the temperature dependence of catalytic rates

Abstract

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 warmactive 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 coldactive 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.enzyme cold adaptation | thermodynamic activation parameters | empirical valence bond | temperature dependence | molecular dynamics C old-adapted enzymes are able to catalyze their reactions with chemical rates comparable to and often exceeding those of their warm-active counterparts, and they are a remarkable example of how evolution can tune the biophysical properties of enzymes. Despite major efforts in the last few decades, the structural mechanisms responsible for low-temperature activity still remain largely unknown. A key problem with lowering the temperature is the exponential decrease in enzyme reaction rates, which according to transition state theory is given byHere, k rxn is the reaction rate, T the temperature, κ is a transmission coefficient, k and h are Boltzmann's and Planck's constants, respectively, and ΔG ‡ is the free energy of activation. Decreasing the temperature from 37°C to 0°C typically results in a 20-to 250-fold reduction of the activity of a mesophilic enzyme (1). Clearly, survival at low temperatures requires that both enzyme kinetics and protein stability are adapted accordingly.A remarkable and universal feature of reactions catalyzed by cold-adapted enzymes is a lower enthalpy and a more negative entropy of activation compared with thermophilic orthologs (1-3). This enthalpy-entropy phenomenon, as indicated in Fig. 1A, coincides with the fact that psychrophilic enzymes have a reduced thermostability, and thus melt at lower temperatures than their mesophilic counterparts. It is evident from Eq. 1 that a lower activation enthalpy makes the rate less tempe...

Thermodynamic and Structural Adaptation Differences between the Mesophilic and Psychrophilic Lactate Dehydrogenases

“…In our case, this restriction is more profound for the more flexible protein, cgLDH, as expected for the cold-adapted enzyme. 31,32 …”

Temperature adaptation of DNA ligases from psychrophilic organisms

“…Computer simulations and Arrhenius plots suggest that surface rigidity/flexibility outside the catalytic region affects the enthalpy/entropy balance. Key single distant mutations may disrupt surface hydrogen binding networks and alter the protein water surface interactions (Isaksen et al 2016) which may be the case with arginine substitutions in our study.…”

“…EM introduces a reversible inactivated (not denatured) form of the enzyme (Einact) as an intermediate in rapid equilibrium with the active form (Eact), which adds a thermal buffer effect that protects the enzyme from thermal inactivation. Another explanation invokes a tuning of surface mobility through alteration of regions spatially removed from the active site which affect the overall enzyme dynamics (Åqvist et al 2017;Isaksen et al 2016). Computer simulations and Arrhenius plots suggest that surface rigidity/flexibility outside the catalytic region affects the enthalpy/entropy balance.…”

“…It is now widely accepted that structural differences between cold-active enzymes and their mesophilic counterparts enable high specific activity at low temperatures, with a lower energy cost (D'Amico et al 2002;Feller 2003;Struvay and Feller 2012). The physical origin of decreased temperature optima imparted by these structural changes are an active area of contemporary investigation (Åqvist et al 2017;Arcus et al 2016;Isaksen et al 2016;Saavedra et al 2018;van der Kamp et al 2018), but it is generally observed that improved catalytic efficiency is accompanied by a reduced thermal stability and weaker substrate affinity, compared to thermophiles and mesophiles at the opposite end of the temperature scale (Struvay and Feller 2012). For this reason we have also carried out in silico comparisons of these Arctic-derived ADLs with mesophilic-derived counterparts from human pathogens.…”

Investigation on Thermodynamic Variables of the Enzyme During the Cryotherapy

“…the lower ∆ ⧧ makes the rate less temperature dependent, and the smaller the activation enthalpy corresponding to more negative value of ∆ ⧧ which means flexibility of the active site [11].…”