Single-Molecule Sampling of Dihydrofolate Reductase Shows Kinetic Pauses and an Endosteric Effect Linked to Catalysis

Abstract

The ability to sample multiple reactions on the same single enzyme is important to link rare intermediates with catalysis and to unravel the role of conformational changes. Despite decades of efforts, however, the single-molecule characterization of nonfluorogenic enzymes during multiple catalytic turnovers has been elusive. Here, we show that nanopore currents allow sampling the dynamic exchange between five structural intermediates during E. coli dihydrofolate reductase (DHFR) catalysis. We found that an endosteric effect promotes the binding of the substrate to the enzyme with a specific hierarchy. The chemical step then switched the enzyme from the closed to the occluded conformation, which in turn promotes the release of the reduced cofactor NADP + . Unexpectedly, only a few reactive complexes lead to catalysis. Furthermore, second-long catalytic pauses were observed, possibly reflecting an off-path conformation generated during the reaction. Finally, the free energy from multiple cofactor binding events were required to release the product and switch DHFR back to the reactive conformer. This catalytic fueled concerted mechanism is likely to have evolved to improve the catalytic efficiency of DHFR under the high concentrations of NADP + in E. coli and might be a general feature for complex enzymatic reactions where the binding and release of the products must be tightly controlled.

Nanopore-based technologies beyond DNA sequencing

“…Ligand-induced conformational changes for a range of proteins [78][79][80] have been reported using biological nanopores. These include the tiny conformational changes of dihydrofolate reductase (DHFR) during ligand binding 80 and catalysis 81 , which could not be observed previously by single-molecule fluorescence resonance energy transfer experiments. The results of these studies revealed that DHFR exists in multiple fixed conformations-conformers-whose exchange during catalysis is probably used to tune enzyme efficiency 80,81 .…”

“…d, Catalytic activity of DHFR (coloured according to vacuum electrostatics using PyMOL) inside a ClyA nanopore (grey, top), as shown by representative traces (bottom). Product formation is indicated by pink asterisks81 , which involve the complexes of the enzyme DHFR (E) with NADPH (E:NH), tetrahydrofolate (E:THF) and then dihydrofolate (E:NH:DHF). e, Dynamic conformation of a single peptide confined in a SiN x solid-state nanopore (top left).…”

“…Springer Nature Ltd; c, ref 68 ,. American Chemical Society; d, ref 81 ,. American Chemical Society; e, ref 83 ,.…”

Application of Nanopore Sensors for Biomolecular Interactions and Drug Discovery

“…Furthermore, by sampling multiple reactions of a single enzyme using ClyA mutant pores (Figure 1B), nanopore currents reveal the conformational dynamics between five intermediate structures during catalysis, and the enzyme reaction can be observed by nanopore sensing at the singlemolecule level. [23] In 2022, Li et al used ClyA-AS mutant nanopores to investigate the morphological changes in the Abelson (Abl) [19] Copyright 2015, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. (B) Trapping of DHFR with ClyA-AS nanopore, providing distinctive current blockades by transition state responding to DHFR catalytic cycle.…”

“…Reproduced with permission. [23] Copyright 2022 The Authors. Published by American Chemical Society.…”