Dihydrofolate reductase (DHFR) reduces dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. Due to its role in one carbon metabolism, chromosomal DHFR is the target of the antibacterial drug, trimethoprim. Bacteria have evolved a type II DHFR that is not structurally related to the chromosomal enzyme to resist trimethoprim. Previous studies examined the effects of small molecule osmolytes on the substrate interactions using two non-homologous DHFRs, E. coli chromosomal DHFR (EcDHFR) and R67 DHFR, which have vastly different active site structures. The results indicated that DHF weakly interacts with the osmolytes in solution, shifting the binding equilibrium from bound to unbound DHF. It is hypothesized that similar weak, nonspecific interactions (quinary interactions) may also occur between DHF and macromolecules that are typically used to crowd solutions (crowders). Because of its metabolic significance, understanding DHFR kinetics and ligand binding behavior in more cell-like conditions is important. The kinetics and ligand binding properties of EcDHFR and R67 DHFR were studied in the presence of macromolecular crowders. There were varied effects on NADPH oxidation and binding to the two DHFRs, with some crowders increasing affinity and others weakening binding. However, DHF binding and reduction by both DHFRs decreased in the presence of all crowders. The decreased binding of ligands is mostly attributed to quinary interactions with the macromolecules, as opposed to crowder effects on the DHFRs. The net charge of protein crowders correlated with effects on NADP+ binding, with near neutral and positively charged proteins having more detrimental effects. For DHF, effects on binding correlated more with the size of binding pockets on the protein crowders. These non-specific interactions between DHFR ligands and proteins predict that the in vivo efficiency of DHFRs may be much lower than expected from their in vitro rates in dilute solutions.

Macromolecular crowders weaken ligand binding to dihydrofolate reductases through quinary interactions

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.