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

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Abstract
To characterize the thermal stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, urea-induced unfolding of 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 ΔΔG° of each unfolding process. Other thermodynamic parameters, enthalpy change ΔΔH, entropy change ΔΔS and heat capacity change ΔΔCp, were derived from the temperature dependence of ΔΔG°. The main feature of the thermophilic enzyme was its lower dependence of ΔΔG° on temperature resulting from a low ΔΔCp. The thermophilic IPMDH had a significantly lower ΔΔCp, 1.73 kcal/mol.K, than that of E.coli IPMDH (20.7 kcal/mol.K). The low ΔΔCp of T.thermophilus IPMDH could not be predicted from its change in solvent-accessible surface area ΔΔASA. 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 Ts. The ΔΔG° maximum of T.thermophilus IPMDH was much smaller than that of E.coli IPMDH. The present results clearly demonstrated that a higher melting temperature Tm is not necessarily accompanied by a higher ΔΔG° maximum.