Interaction of 13C-enriched folate with dihydrofolate reductase studied by carbon magnetic resonance spectroscopy

In the presence of dihydrofolate reductase the carbon magnetic resonance spectrum of folate labeled at the benzoylcarbonyl carbon with ¹³C contains two broadened peaks arising from free and enzyme-bound folate, the latter appearing over 2 ppm upfield from free folate. Addition of TPN⁺ causes sharpening of both peaks indicating formation of a single folate-TPN⁺-enzyme ternary complex. Methotrexate specifically displaces folate from the ternary complex regenerating a single sharp resonance at 170.4 ppm characteristic of free folate. Line width changes show that folate is bound more tightly in the ternary than in the binary complex. Increased shielding of this carbonyl upon binding is inconsistent with its participation in a H-bond.