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 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 (<DeltaH(binding)> = -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-oxo-dGMP to MutT induces changes in backbone (15)N 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.
Saraswat
Vibhor
V
Department of Biological Chemistry, The Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2185, USA.
Massiah
Michael A
MA
Lopez
Gregory
G
Amzel
L Mario
LM
Mildvan
Albert S
AS
D002151:Calorimetry; D002413:Cations, Divalent / Q000737:chemistry; D003848:Deoxyguanine Nucleotides / Q000737:chemistry*; D011756:Diphosphates / Q000737:chemistry*; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives* / Q000737:chemistry*; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008956:Models, Chemical; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011522:Protons; D011755:Pyrophosphatases; D013696:Temperature; D013816:Thermodynamics
D002413:Cations, Divalent; D003848:Deoxyguanine Nucleotides; D011756:Diphosphates; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D009587:Nitrogen Isotopes; D011522:Protons; C078206:8-oxodeoxyguanosine triphosphate; D008345:Manganese; C007257:2'-deoxyguanosine 5'-phosphate; D006157:Guanosine Monophosphate; C039543:8-oxoguanosine 2'-phosphate; D010744:Phosphoric Monoester Hydrolases; D011755:Pyrophosphatases; C052211:mutT protein, E coli; D008274:Magnesium