Spin probes as mechanistic inhibitors and active site probes of thymidylate synthetase

C-4- and C-5-substituted analogues of dUMP were examined as inhibitors of thymidylate synthetase and as topographical probes of its active site by electron spin resonance (ESR). The C-5-substituted spin-labeled analogues pDUAP (2) and a pDUTT (3) as well as the unlabeled AAdUMP (1) were competitive inhibitors with Ki's of 9.2, 89, and 7.9 microM, respectively. The C-4-spin-labeled pls4dU (4) displayed no inhibition activity. Scatchard plots as determined by ESR gave similar association constants for 2 (Kassoc = 1.9 X 10(5) M-1) and for 3 (Kassoc = 2.4 X 10(5) M-1). Both of these values are similar to the Kassoc of FdUMP indicating that the bulky substituent in position 5 does not interfere with the formation of the binary complex. The enzyme-C-5-spin-labeled nucleotide complexes indicate the presence of similarly immobilized spin labels by ESR, whereas no binding and immobilization were noticed with the C-4-spin-labeled nucleotide. A model for the active-site geometry of the enzyme was derived which suggests that the C-5 substituents point toward the opening of the binding cavity whose depth is at least 12 A. Also, the approximate 10-fold increased inhibitory activity of 2 as compared to that of 3 may be attributed to the significant electron withdrawing properties of the C-5 substituent in 2. Finally, the set of probes used for the binding and inhibition of thymidylate synthetase gives direct experimental evidence that an electron-withdrawing C-5 substituent primarily affects the formation of the ternary complex and will not substantially influence the stability of the binary complex.