Synergism in folding of a double mutant of the alpha subunit of tryptophan synthase.

The urea-induced unfolding of the inactive single mutants Tyr-175----Cys and Gly-211----Glu and the active double mutant Cys-175/Glu-211 of the alpha subunit of tryptophan synthase from Escherichia coli was examined by using ultraviolet difference spectroscopy. Equilibrium techniques were used to determine the equilibrium free energies of unfolding for the mutant proteins to permit comparison with the wild-type protein. The sum of the changes in stability for the single mutants is not equal to the change seen in the double mutant. This inequality is evidence for a structural interaction between these two residues. Kinetic studies show that this synergism, which destabilizes the native form by 1.5-2.0 kcal/mol at pH 7.8, 25 degrees C, occurs only after the final rate-limiting step of domain association.