High thermal stability of 3-isopropylmalate dehydrogenase from Thermus thermophilus resulting from low ΔCp of unfolding

To characterize the thermal stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, urea-induced unfolding off the enzyme and of its mesophilic counterpart from Escherichia coli was investigated at various temperatures. The unfolding curves were analyzed with a three,state model for E.coli IPMDH and with a two-state model for T. thermophilus IPMDH, to obtain the free energy change DeltaGdegrees of each unfolding process. Other thermodynamic parameters, enthalpy change DeltaH , entropy change DeltaS and heat capacity change DeltaC(p), were derived from the temperature dependence of DeltaGdegrees. The main feature of the thermophilic enzyme was its lower dependence of DeltaGdegrees on temperature resulting from a low ODelta(p). The thermophilic IPMDH had a significantly lower DeltaC(p), 1.73 kcal/mol.K, than that of E.coli IPMDH (20.7 kcal/mol.K). The low DeltaC(p) of T. thermophilus IPMDH could not be predicted from its change in solvent-accessible surface area DeltaASA. The results suggested that there is a large structural difference between the unfolded state of T. thermophilus and that of E.coli IPMDH. Another responsible factor for the higher thermal stability of T. thermophilus IPMDH was the increase in the most stable temperature T-s. The DeltaGdegrees maximum of T. thermophilus IPMDH was much smaller than that of E.coli IPMDH. The present results clearly demonstrated that a higher melting temperature T-m is not necessarily accompanied by a higher DeltaGdegrees maximum.