Substrate binding and turnover modulate the affinity landscape of dihydrofolate reductase to increase its catalytic efficiency

It is generally accepted that enzymes structures evolved to stabilize the transition-state of a catalyzed reaction. Here, observing single molecules with a multi-turnover resolution, we provide experimental evidence for a more sophisticated narrative. We found that the binding of the NADPH cofactor to DHFR induces a first allosteric change that increases the affinity of the enzyme for the substrate. Then the enthalpy generated by the chemical step provides a power stroke that switches the enzyme to the product-bound conformations and promotes the release of the oxidized cofactor NADP+. The subsequent binding of NADPH to the vacated site provides the free energy for the recovery stroke, which induces the allosteric release of the product and resets the initial configuration. Intriguingly, the cycle is not perfect. Occasionally, DHFR undergoes second-long catalytic pauses, most likely reflecting the occupancy of an off-path conformation induced by excess energy liberated by the chemical step. This catalytic remodeling of the affinity landscape of DHFR is likely to have evolved to improve the efficiency of the reaction to cope with the high concentration of NADP+ in E. coli. And might be a general feature for complex enzymatic reaction where the binding and release of the products must be tightly controlled.