MODE OF BINDING OF FOLATE ANALOGS TO THYMIDYLATE SYNTHASE - EVIDENCE FOR 2 ASYMMETRIC BUT INTERACTIVE SUBSTRATE-BINDING SITES

Human thymidylate synthase is a polymeric protein composed of two subu nits with identical primary structures. In this study we determined th e binding affinities of 5,10-methylene tetrahydropteroyltetraglutamate (folate substrate) and a group of close structural folate analog inhi bitors. Thymidylate synthase bound both mono and polyglutamylated fola te substrates and analogs more tightly in the presence of deoxyuridyla te. These results and product inhibition studies confirmed that the or ders of substrate addition and product release from thymidylate syntha se were similar for mono and polyglutamylated substrates. Equilibrium dialysis studies showed that the folate substrate in a ternary complex with deoxyuridylate bound to one of the subunits (site A) with a K(d) of 720 nm. The binding of the substrate to the second subunit (site B ) was much weaker, and the K(d) could not be determined by this method . However, dissociation constants for each subunit could be measured f or the folate analog inhibitors, and, depending on the inhibitor, the relative K(d) value for each subunit varied substantially. For example , formyl-5,8-dideazafolate and tetraglutamylated 10-propargyl-5,8-dide azafolate bound to both sites with similar K(d) values, whereas D1694G lu4 bound to subunit A with a higher affinity (K(d) = 1.0 nM) than to subunit B (K(d) = 30 nM). In contrast, 1843U89 (mono or diglutamylated form) had a much higher affinity for subunit B (K(d) approximately 1 nM) compared with subunit A (K(d) approximately 400 nM). Enzyme inhibi tion kinetic analyses showed that the K(i) values of 1843U89 were quit e low (0.1 nM) and that the inhibition was noncompetitive. In contrast , the other folate analogs inhibited the enzyme via mixed inhibition ( i.e. both the K(m) for the folate substrate and the V(max) were altere d). We conclude that the two subunits of thymidylate synthase bind fol ate substrates and analogs differently and that the asymmetric binding of the ligands is the major factor that determines the inhibition kin etics of each folate analog inhibitor.