Thermodynamic framework for identifying free energy inventories of enzyme catalytic cycles.

Pauling's suggestion that enzymes are complementary in structure to the activated complexes of the reactions they catalyze has provided the conceptual basis to explain how enzymes obtain their fantastic catalytic prowess, and has served as a guiding principle in drug design for over 50 y. However, this model by itself fails to predict the magnitude of enzymes' rate accelerations. We construct a thermodynamic framework that begins with the classic concept of differential binding but invokes additional terms that are needed to account for subtle effects in the catalytic cycle's proton inventory. Although the model presented can be applied generally, this analysis focuses on ketosteroid isomerase (KSI) as an example, where recent experiments along with a large body of kinetic and thermodynamic data have provided strong support for the noncanonical thermodynamic contribution described. The resulting analysis precisely predicts the free energy barrier of KSI's reaction as determined from transition-state theory using only empirical thermodynamic data. This agreement is suggestive that a complete free energy inventory of the KSI catalytic cycle has been identified.