Thermodynamics and kinetics of folding of common-type acylphosphatase: Comparison to the highly homologous muscle isoenzyme

The thermodynamics and kinetics of folding of common-type acylphosphatase h ave been studied under a variety of experimental conditions and compared wi th those of the homologous muscle acylphosphatase. Intrinsic fluorescence a nd circular dichroism have been used as spectroscopic probes to follow the folding and unfolding reactions. Both proteins appear to fold via a two-sta te mechanism. Under all the conditions studied, common-type acylphosphatase possesses a lower conformational stability than the muscle form. Neverthel ess, common-type acylphosphatase folds more rapidly, suggesting that the co nformational stability and the folding rate are not correlated in contrast to recent observations for a number of other proteins. The unfolding rate o f common-type acylphosphatase is much higher than that of the muscle enzyme , indicating that the differences in conformational stability between the t wo proteins are primarily determined by differences in the rate of unfoldin g. The equilibrium m value is markedly different for the two proteins in th e pH range of maximum conformational stability (5.0-7.5); above pH 8.0, the m value for common-type acylphosphatase decreases abruptly and becomes sim ilar to that of the muscle enzyme, Moreover, at pH 9.2, the dependencies of the folding and unfolding rate constants of common-type acylphosphatase on denaturant concentration (mf and m, values, respectively) are notably redu ced with respect to pH 5.5. The pH-induced decrease of the m value can be a ttributed to the deprotonation of three histidine residues that are present only in the common-type isoenzyme, This would decrease the positive net ch arge of the protein, leading to a greater compactness of the denatured stat e. The folding and unfolding rates of common-type acylphosphatase are not, however, significantly different at pH 5.5 and 9.2, indicating that this ch ange in compactness of the denatured and transition states does not have a notable influence on the rate of protein folding.