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Inhibition of R67 DHFR activity (51 μM NADPH and 4–56 μM DHF) with folate derivatives in 10 mM Tris-HCl, 100 mM NaCl, and 1 mM EDTA (pH 7.0) at 25 °C. (A) Competitive inhibition of R67 by folate yields a Ki of 16 ± 3 μM. Folate concentrations are 0 μM (—), 13.5 μM (−–−), 26 μM (···), and 62 μM (−-−). (B) The Bis-EDA–folate adduct inhibits R67 DHFR in a noncompetitive manner (Ki = 140 ± 15 μM). Bis-EDA–folate adduct concentrations are 0 μM (—), 50 μM (−–−), and 100 μM (···).
