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Interactions of the Products, 8-Oxo-dGMP, dGMP, and Pyrophosphate with the MutT Nucleoside Triphosphate Pyrophosphohydrolase

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journal contribution
posted on 05.12.2002 by Vibhor Saraswat, Michael A. Massiah, Gregory Lopez, L. Mario Amzel, Albert S. Mildvan
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 103.4-fold lower Km 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 KIslope = 49 nM, which is 104.6-fold lower than the KIslopeof dGMP (1.7 mM). Similarly, the KIintercept of 8-oxo-dGMP is 104.0-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 KD = 1.8 mM, in agreement with its KIslope. With 8-oxo-dGMP, tight binding and slow exchange (n = 1.0 ± 0.1, KD < 0.25 mM) are found. Isothermal calorimetric titration of MutT with 8-oxo-dGMP yields a KD of 52 nM, in agreement with its KIslope. Changing the metal activator from Mg2+ to Mn2+ had little effect on the KIslope of dGMP or of 8-oxo-dGMP, consistent with the second-sphere 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 KI 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 (〈ΔHbinding〉 = −32 ± 7 kcal/mol), with an unfavorable entropy (〈− 〉 = +22 ± 7 kcal/mol), as determined by van't Hoff analysis of the effect of temperature on the KIslope 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.