Thermodynamic properties of the transition state for the rate-limiting step in the folding of the alpha subunit of tryptophan synthase.

To gain insight into the physical properties of the transition state for the rate-limiting step in the folding of the alpha subunit of tryptophan synthase from Escherichia coli, the urea dependence of the unfolding reaction was examined as a function of temperature. Consistent with a previous, more limited study [Hurle, M.R., Michelotti, G.A., Crisanti, M.M., & Matthews, C.R. (1987) Proteins 2, 54], the activation entropy for unfolding was found to be negative above 4 M urea. The present study extends this finding to show that both the activation entropy and enthalpy decrease with increasing urea concentrations between 4 and 7.5 M. The change in the heat capacity from the native to the transition state is positive and appears to increase with the denaturant concentration. The urea and temperature dependences of the unfolding rates were analyzed in terms of the denaturant-binding model of Tanford [Tanford, C. (1970) Adv. Protein Chem. 24, 1]. The values for the activation enthalpy and activation entropy of binding are in good agreement with those obtained from a calorimetric study of urea binding to unfolded proteins [Makhatadze, G.I., & Privalov, P.L. (1992) J. Mol. Biol. 226, 491]. These results show that (1) the binding of urea to the transition state of the alpha subunit has thermodynamic properties which are similar to those for urea binding to unfolded proteins, (2) the transition state is distinct from the unfolded conformation and exposes only a fraction of its urea-binding sites to solvent, and (3) the negative value for the activation entropy for unfolding reflects, in part, the ordering of urea on newly exposed surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)