pyrophosphate and 2--deoxyguanosine-5--phosphate

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 Pbeta, 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 Mg(2+)-activated dGTP hydrolysis, 8-oxo-dGMP is a noncompetitive inhibitor with K(I)(sl)(o)(pe) = 49 nM, which is 10(4.6)-fold lower than the K(I)(sl)(o)(pe)of dGMP (1.7 mM). Similarly, the K(I)(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, (1)H-(15)N 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 K(I)(sl)(o)(pe). 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 K(I)(sl)(o)(pe). Changing the metal activator from Mg(2+) to Mn(2+) had little effect on the K(I)(sl)(o)(pe) of dGMP or of 8-oxo-dGMP, consistent with the second-sphere enzyme-M(2+)-H(2)O-NTP-M(2+) 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 K(I) 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 (DeltaG degrees = -9.8 kcal/mol) is driven by a highly favorable enthalpy ( = -32 +/- 7 kcal/mol), with an unfavorable entropy (<-TDeltaS(o)(binding)> = +22 +/- 7 kcal/mol), as determined by van't Hoff analysis of the effect of temperature on the K(I)(sl)(o)(pe) and by isothermal titration calorimetry in two buffer systems. The binding of 8-o

Topics: Calorimetry; Cations, Divalent; Deoxyguanine Nucleotides; Diphosphates; Enzyme Activation; Enzyme Activators; Enzyme Inhibitors; Escherichia coli Proteins; Guanosine Monophosphate; Kinetics; Macromolecular Substances; Magnesium; Manganese; Models, Chemical; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular; Phosphoric Monoester Hydrolases; Protons; Pyrophosphatases; Temperature; Thermodynamics

The MutT protein, which prevents AT----CG transversions during DNA replication, hydrolyzes nucleoside triphosphates to yield nucleoside monophosphates and pyrophosphate. The hydrolysis of dGTP by the MutT protein in H(2)18O-enriched water, when monitored by high resolution 31P NMR spectroscopy at 242.9 MHz, showed 18O labeling of the pyrophosphate product, as manifested by a 0.010 +/- 0.002 ppm upfield shift of the pyrophosphate resonance, and no labeling of the dGMP product. This establishes that the reaction proceeds via a nucleophilic substitution at the beta-phosphorus of dGTP with displacement of dGMP as the leaving group. No exchange of 32P-labeled dGMP into dGTP was detected, indicating that water attacks dGTP directly or, less likely, an irreversibly formed pyrophosphoryl-enzyme intermediate. No exchange of 32P-labeled pyrophosphate into dGTP was observed, consistent with nucleophilic substitution at the beta-phosphorus of dGTP. Only six enzymes, all synthetases, have previously been shown to catalyze nucleophilic substitution at the beta-phosphorus of nucleoside triphosphate substrates. The MutT protein is the first hydrolase shown to do so.

Topics: Bacterial Proteins; Deoxyguanine Nucleotides; Diphosphates; DNA Repair; Escherichia coli; Escherichia coli Proteins; Hydrolysis; Isotope Labeling; Magnetic Resonance Spectroscopy; Oxygen Isotopes; Phosphoric Monoester Hydrolases; Pyrophosphatases; Water