Abstract
Structures of enzymes invariably reveal the proximity of acidic and basic residues to reactive sites on the substrate, so it is natural and common to suggest that enzymes employ concerted mechanisms to catalyze their difficult reactions. Ketosteroid isomerase (KSI) has served as a paradigm of enzymatic proton transfer chemistry, and its catalytic effect has previously been attributed to concerted proton transfer. We employ a specific inhibitor that contains an IR probe that reports directly and quantitatively on the ionization state of the ligand when bound in the active site of KSI. Measurement of the fractional ionization provides a missing link in a thermodynamic cycle that can discriminate the free energy advantage of a concerted versus nonconcerted mechanism. It is found that the maximum thermodynamic advantage that KSI could capture from a concerted mechanism (ΔΔG° = 0.5 kcal mol(-1)) is quite small.