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The binding of substrates and inhibitors to dihydrofolate reductase was studied by steady-state kinetics and high-field 1H-n.m.r. spectroscopy. A series of 5-substituted 2,4-diaminopyrimidines were examined and were found to be 'tightly binding' inhibitors of the enzyme (Ki less than 10(-9) M). Studies on the binding of 4-substituted benzenesulphonamides and benzenesulphonic acids also established the existence of a 'sulphonamide-binding site' on the enzyme. Subsequent n.m.r. experiments showed that there are two binding sites for the sulphonamides on the enzyme, one of which overlaps the coenzyme (NADPH) adenine-ring-binding site. An examination of the pH-dependence of the binding of sulphonamides to the enzyme indicated the influence of an ionizable group on the enzyme that was not directly involved in the sulphonamide binding. The change in pKa value from 6.7 to 7.2 observed on sulphonamide binding suggests the involvement of a histidine residue, which could be histidine-28.

Mouse fibroblasts (strain L) resistant to growth inhibition by 2,6-diaminopurine (DAP) were obtained from DAP-sensitive cultures. DAP-resistant cells were characterized by (a) stability of resistance in the absence of DAP, (b) cross resistance to 8-azaadenine, (c) very limited utilization of C14-labelled DAP or adenine for the synthesis of nucleic acid purines, and (d) greatly decreased adenylic acid and DAP ribonucleotide pyrophosphorylase activities. The base ratios for ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) of DAP-resistant cells did not differ significantly from the corresponding ratios for DAP-sensitive cells.The metabolism of DAP by L-cells was found to be similar to the metabolism of the compound by rodent tissues in vivo. In the presence of low concentrations of DAP-2-C14, DAP-sensitive L-cells readily utilized DAP for the synthesis of acid-soluble derivatives and nucleic acid purines, especially guanine. At low concentrations of DAP-2-C14the utilization of DAP-2-C14by DAP-resistant and DAP-sensitive cells increased with the concentration of DAP-2-C14, but the utilization of DAP-2-C14by DAP-resistant cells increased at a very much lower rate than the utilization of the analogue by DAP-sensitive cells. Adenine-C14was utilized more extensively than DAP-2-C14for the synthesis of nucleic acids of DAP-resistant cells. No correlation between the intracellular concentration of any acid-soluble derivative of DAP and inhibition of cell growth was found.