Relationship between functional activity and protein stability in the presence of all classes of stabilizing osmolytes

We report the effects of stabilizing osmolytes (low molecular mass organic compounds that raise the midpoint of thermal denaturation) on the stability and function of RNase-A under physiological conditions (pH 6.0 and 25 degrees C). Measurements of Gibbs free energy change at 25 degrees C (delta G(D)degrees) and kinetic parameters, Michaelis constant (K-m) and catalytic constant (k(cat)) of the enzyme mediated hydrolysis of cytidine monophosphate, enabled us to classify stabilizing osmolytes into three different classes based on their effects on kinetic parameters and protein stability. (a) Polyhydric alcohols and amino acids and their derivatives do not have significant effects on delta G(D)degrees and functional activity (K-m and k(cat)). (b) Methylamines increase delta G(D)degrees and k(cat), but decrease K-m. (c) Sugars increase delta G(D)degrees, but decrease both K-m and k(cat). These findings suggest that, among the stabilizing osmolytes, (a) polyols, amino acids and amino acid derivatives are compatible solutes in terms of both stability and function, (b) methylamines are the best refolders (stabilizers), and (c) sugar osmolytes stabilize the protein, but they apparently do not yield functionally active folded molecules.