Interactions of the products, 8-Oxo-dGMP, dGMP, and pyrophosphate with the MutT nucleoside triphosphate pyrophosphohydrolase

The MutT enzyme from E. coli, in the presence of a divalent cation, catalyzes the hydrolysis of nucleoside- and deoxynucleoside-triphosphate (NTP) substrates by nucleophilic substitution at Pβ, to yield a nucleotide (NMP) and PPi. The best substrate of MutT is believed to be the mutagenic nucleotide 8-oxo-dGTP, on the basis of its 10^3.4-fold lower K_m than that of dGTP (Maki, H., and Sekiguchi, M. (1992) Nature 355, 273-275). To determine the true affinity of MutT for an 8-oxo-nucleotide and to elucidate the kinetic scheme, product inhibition by 8-oxo-dGMP and dGMP and direct binding of these nucleotides to MutT were studied. With Mg²⁺-activated dGTP hydrolysis, 8-oxo-dGMP is a noncompetitive inhibitor with K₁^slope = 49 nM, which is 10^4.6-fold lower than the K₁^slope of dGMP (1.7 mM). Similarly, the K₁^intercept of 8-oxo-dGMP is 10^4.0-fold lower than that of dGMP. PPi is a linear uncompetitive inhibitor, suggesting that it dissociates first from the product complex, followed by the nucleotide. Noncompetitive inhibition by dGMP and 8-oxo-dGMP indicates an "iso" mechanism in which the nucleotide product leaves an altered form of the enzyme which slowly reverts to the form which binds substrate. Consistent with this kinetic scheme, ¹H-¹⁵N HSQC titration of MutT with dGMP reveals weak binding and fast exchange from one site with a K_D = 1.8 mM, in agreement with its K₁^slope. With 8-oxo-dGMP, tight binding and slow exchange (n = 1.0 ± 0.1, K_D < 0.25 mM) are found. Isothermal calorimetric titration of MutT with 8-oxo-dGMP yields a K_D of 52 nM, in agreement with its K₁^slope. Changing the metal activator from Mg²⁺ to Mn²⁺ had little effect on the K₁^slope of dGMP or of 8-oxo-dGMP, consistent with the secondsphere enzyme - M²⁺-H₂O-NTP - M²⁺ complex found by NMR (Lin, J., Abeygunawardana, C., Frick, D. N., Bessman, M. J., and Mildvan, A. S. (1997) Biochemistry 36, 1199 -1211), but it decreased the K₁ of PPi 12-fold, suggesting direct coordination of the PPi product by the enzyme-bound divalent cation. The tight binding of 8-oxo-dGMP to MutT (ΔG° = -9.8 kcal/mol) is driven by a highly favorable enthalpy (〈ΔH_binding〉 = -32 ± 7 kcal/mol), with an unfavorable entropy (〈-TΔS°_binding〉 = +22 ± 7 kcal/mol), as determined by van't Hoff analysis of the effect of temperature on the K₁^slope and by isothermal titration calorimetry in two buffer systems. The binding of 8-oxo-dGMP to MutT induces changes in backbone ¹⁵N and NH chemical shifts of 62 residues widely distributed throughout the protein, while dGMP binding induces smaller changes in only 22 residues surrounding the nucleotide binding site, suggesting that the unusually high affinity of MutT for 8-oxo-nucleotides is due not only to interactions with the altered 8-oxo or 7-NH positions on guanine, but results primarily from diffuse structural changes which tighten the protein structure around the 8-oxo-nucleotide.