High thermal stability of 3-isopropylmalate dehydrogenase from Thermus thermophilus resulting from low DeltaC(p) of unfolding.

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 DeltaG degrees 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 DeltaG degrees. The main feature of the thermophilic enzyme was its lower dependence of DeltaG degrees on temperature resulting from a low DeltaC(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 DeltaG degrees 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 DeltaG degrees maximum.

Urea-induced unfolding and conformational stability of 3-isopropylmalate dehydrogenase from the Thermophile thermus thermophilus and its mesophilic counterpart from Escherichia coli.

To reveal the basis of 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 has been studied. The urea-induced equilibrium unfolding of T. thermophilus and E. coli IPMDHs at 27 degreesC was monitored by measuring the changes in far-UV CD, intrinsic fluorescence, anilinonaphthalenesulfonic acid (ANS) binding, and catalytic activity in the presence of nonionic detergent Tween 20. For both enzymes, the spectral methods revealed a biphasic unfolding transition. The first transition was protein concentration-independent, whereas the second was protein concentration-dependent for both enzymes. The observation suggested a three-state unfolding mechanism with a dimeric intermediate. However, the intermediates of the E. coli and the T. thermophilus IPMDHs seemed to be different from each other. The intermediate of the E. coli IPMDH lost its secondary and tertiary structure more than that of the thermophilic enzyme. E. coli IPMDH lost enzymatic activity through the transition from the native to the intermediate state, though the intermediate of the T. thermophilus enzyme was still active. The unfolding process of E. coli IPMDH can be explained by a sequential unfolding of individual folding domains, while there is only a small structural perturbation in the intermediate of T. thermophilus IPMDH. The higher thermal stability of T. thermophilus IPMDH can be attributed to the increase in the extent of interaction inside the first domain which unfolded prior to the unfolding of the whole molecular structure in E. coli IPMDH.