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

Abstract

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 offpath 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.

Nanopores: a versatile tool to study protein dynamics

“…Hel 308 (cyan), detected using the MspA protein pore (yellow) as steps in the recorded nanopore current [97]. B) The functional cycle of dihydrofolate reductase (DHFR) directly observed by electroosmotic trapping in the ClyA protein pore sensor resolves five functional states (four displayed) [98].…”

The NEOtrap – en route with a new single-molecule technique

“…It will be interesting to see what the limits are for resolving conformational transitions with the NEOtrap, which – unlike FRET – is not limited to one specific reaction coordinate. Indeed, nanopore detection has already revealed hundreds of catalytic cycles of a single enzyme that were undetectable by FRET ( Galenkamp and Maglia, 2020 ). The label-free study of diverse dynamic protein systems in the range of microseconds to minutes bears the potential to reveal hierarchical correlations between fast and slow transitions that were missed with existing techniques that sample either the fast or the slow end of the time axis.…”

Automated Electrical Quantification of Vitamin B1 in a Bodily Fluid using an Engineered Nanopore Sensor

“…[44][45][46][47][48] Interestingly,t he environment of these large nanopores is not dissimilar from bulk, [49] and allows to perform single-molecule enzymological studies. [50][51][52] Nanopores can measure small molecules directly with high sensitivity.H owever,e ven though rich electrical signals can be obtained through nanopore measurements, [53,54] the lack of selectivity complicates the identification of small molecules in biological samples.S ubstrate binding proteins can be highly selective.T hus,t he combination of sensitivity of an anopore with the selectivity of as ubstrate binding protein allows the detection and quantification of metabolites in complex samples. [44] Here we use the periplasmic thiamine binding protein (TbpA, Figure 1B), which has as pecific binding affinity to thiamine (Figure 1C).…”