Direct Brønsted analysis of the restoration of activity to a mutant enzyme by exogenous amines

A true Brønsted analysis of proton transfer in an enzyme mechanism is made possible by the chemical rescue of an inactive mutant of aspartate aminotransferase, where the endogenous general base, Lys²⁵⁸, is replaced with Ala by site-directed mutagenesis. Catalytic activity is restored to this inactive mutant by exogenous amines. The eleven amines studied generate a Brønsted correlation with β of 0.4 for the transamination of cysteine suilfinate, when steric effects are included in the regression analysis. Localized mutagenesis thus allows the classical Brønsted analysis of transition-state structure to be applied to enzyme-catalyzed reactions.