Comparing the thermodynamic stabilities of a related thermophilic and mesophilic enzyme

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Abstract

Several models have been proposed to explain the high temperatures required to denature enzymes from thermophilic organisms; some involve greater maximum thermodynamic stability for the thermophile, and others do not. To test these models, we reversibly melted two analogous protein domains in a two-state manner. E2(cd) is the isolated catalytic domain of cellulase E2 from the thermophile Thermomonospora fusca. CenA(P30) is the analogous domain of the cellulase CenA from the mesophile Cellulomonas fimi. When reversibly denatured in a common buffer, the thermophilic enzyme E2(cd) had a temperature of melting (Tm) of 72.2 °C, a van't Hoff enthalpy of unfolding (ΔH(VH)) of 190 kcal/mol, and an entropy of unfolding (ΔS(u)) of 0.55 kcal/(mol·K); the mesophilic enzyme CenA(P30) had a T(m) of 56.4 °C, a Δ(VH) of 107 kcal/mol, and a ΔS(u) of 0.32 kcal/(mol·K). The higher ΔH(VH) and ΔS(u) values for E2(cd) suggest that its free energy of unfolding (ΔG(u)) has a steeper dependence on temperature at the T(m) than CenA(P30). This result supports models that predict a greater maximum thermodynamic stability for thermophilic enzymes than for their mesophilic counterparts. This was further explored by urea denaturation. Under reducing conditions at 30 °C, E2(cd) had a concentration of melting (C(m)) of 5.2 M and a ΔG(u) of 11.2 kcal/mol; CenA(P30) had a C(m) of 2.6 M and a ΔG(u) of 4.3 kcal/mol. Under nonreducing conditions, the C(m) and ΔG(u) of CenA(P30) were increased to 4.5 M and 10.8 kcal/mol at 30 °C; the C(m) for E2(cd) was increased to at least 7.4 M at 32 °C. We were unable to determine a ΔG(u) value for E2(cd) under nonreducing conditions due to problems with reversibility. These data suggest that E2(cd) attains its greater thermal stability (ΔT(m) = 15.8 °C) through a greater thermodynamic stability (ΔΔG(u) = 6.9 kcal/mol) compared to its mesophilic analogue CenA(P30).

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Beadle, B. M., Baase, W. A., Wilson, D. B., Gilkes, N. R., & Shoichet, B. K. (1999). Comparing the thermodynamic stabilities of a related thermophilic and mesophilic enzyme. Biochemistry, 38(8), 2570–2576. https://doi.org/10.1021/bi9824902

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