Figure 6.

Comparison of equilibrium binding versus TSR analysis Envisage a catalytic protein interacting with two substrates (or substrate analogs), one exhibiting high-affinity binding (dashed RED line), and the other low-affinity binding (solid BLUE line). Equilibrium binding to the stable Michaelis complex (LEFT, Panels A and B) would produce concentration-dependent saturation of the binding site (Panel B). From the observed affinity difference (ΔK_d) between the two substrates, one can calculate a corresponding difference in binding energy, ΔΔG_S (Panel A), for the two substrates interacting with the stable Michaelis complex at the bottom of the reaction coordinate. In contrast, information on the interaction of substrates at the reaction coordinate peak would require a study of binding to the unstable transition state (RIGHT, Panels C and D). Unfortunately, due to the high energy-level and transient nature and of the transition state (denoted by ‡), the relevant binding experiment (Panel D) is technically impossible. However, TSR analysis allows direct calculation (Equation 9) of the transition state binding energy difference, ΔΔG_b (Panel C, yellow) between two competing substrates, A and B. A change in the TSR phenotype, or Δ(TSR), thus provides evidence for a change in the graphical separation distance, (Panel D, yellow), for the "impossible experiment" on substrate binding to the unstable transition state. Thus, observation of a Δ(TSR) phenotype reflects underlying structural changes that affect binding discrimination between substrates A and B in the transition state, which are of interest because transition state binding interactions create transport catalysis [2–4, 7] by lowering the activation energy, , and increasing k_cat/K_m. In summary, the equilibrium binding experiment depicted on the left does not address catalysis per se, whereas the TSR experiment depicted on the right does.