Dihydrofolate influences the activity of Escherichia coli dihydrofolate reductase synthesised de novo.

The folding of proteins into their native structures is known to depend on molecular chaperones. However, other ligands or cofactors which still require characterisation are also likely to influence protein folding. The intention of this study was to reveal how the folding of an enzyme, Escherichia coli dihydrofolate reductase, was affected by a substrate ligand, i.e. dihydrofolate. The enzyme was synthesised by coupled transcription/translation in a bacterial cell-free system. Correct folding of the protein into its native structure was measured by its enzymatic activity. Synthesis of dihydrofolate reductase was found to be inhibited, at the level of translation, by dihydrofolate. The syntheses of other proteins were also inhibited by this compound and the reasons for this inhibition could not be determined. Most notably, the specific activity of the dihydrofolate reductase formed in the presence of the substrate dihydrofolate was increased and this effect was specific for dihydrofolate reductase since it was not observed with other proteins synthesised in the same system. The increase in dihydrofolate reductase specific activity could not be attributed to mere thermal stabilisation of the fully folded enzyme by dihydrofolate. The effects of dihydrofolate on dihydrofolate reductase synthesis and activity were similar to those of the molecular chaperone DnaJ which is known to promote the folding of newly synthesised proteins. It is suggested that dihydrofolate may interact with the newly synthesised dihydrofolate reductase polypeptide chain and promote its productive folding.