Lawrence Berkeley National Laboratory

A framework that introduces the concept of near-activation complexation and differential free energy of activation is presented to extend the capabilities of the classical transition-state theory in enzymatic reactions. In our approach, reaching a near-equilibrium energy level is assumed to be necessary for complexation near the activation point, whereas an additional differential energy level is required for the near-equilibrium complex to activate and release reaction products. Integration of these energy levels within the transition-state theory explains the thermodynamic nature of the Michaelis–Menten (affinity) constant and its relationship with the rate constant under the quasi-steady-state assumption. The concepts of near-activation complexation and differential free energy of activation were tested on 57 independent experiments of NH+4 and NO-3 uptake by various microalgae and bacteria at temperatures ranging between 1 and 45°C. Results showed that near-activation complexation was always favored, whereas the differential energy of activation led to an apparent energy barrier consistent with earlier observations. Temperature affected all energy levels within this framework but did not alter substantially their thermodynamic features. The approach (1) mutually links the thermodynamics and kinetics of Michaelis-Menten and rate constants with a mathematical expression; (2) describes the likelihood of formation of sub-, super-, and activated complexes; and (3) shows direction and thermodynamic likelihood of each reaction branch within the transition state.