Solution structure, mutagenesis, and NH exchange studies of the MutT enzyme–Mg 2+-8-oxo-dGMP complex

• Published in: Journal of molecular structure Vol. 700; no. 1; pp. 247 - 254
• Main Authors: Massiah, M.A., Saraswat, V., Azurmendi, H.F., Mildvan, A.S.
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 2004
• Subjects: Additive effects; Double mutant; Hydrogen bonds; MutT enzyme; Nucleoside triphosphate; Double mutant; Hydrogen bonds; Additive effects; Nucleoside triphosphate; MutT enzyme
• ISSN: 0022-2860; 1872-8014
• DOI: 10.1016/j.molstruc.2003.12.060

The MutT pyrophosphohydrolase from E. coli (129 residues) catalyzes the hydrolysis of nucleoside triphosphates (NTP), including 8-oxo-dGTP, by substitution at Pβ, to yield NMP and pyrophosphate. The product, 8-oxo-dGMP is an unusually tight binding, slowly exchanging inhibitor with a K D=52 nM, (Δ G°=−9.8 kcal/mol) which is 6.1 kcal/mol tighter than the binding of dGMP (Δ G°=−3.7 kcal/mol). The higher affinity for 8-oxo-dGMP results from a more favorable Δ H binding (−32 kcal/mol) despite an unfavorable − TΔ S° binding (+22 kcal/mol). The solution structure of the MutT–Mg 2+-8-oxo-dGMP complex shows a narrowed, hydrophobic nucleotide-binding cleft with Asn-119 and Arg-78 among the few polar residues. The N119A, N119D, R78K and R78A single mutations, and the R78K+N119A double mutant all showed largely intact active sites, on the basis of small changes in the kinetic parameters of dGTP hydrolysis and in 1H– 15N HSQC spectra. However, the N119A mutation profoundly weakened the active site binding of 8-oxo-dGMP by 4.3 kcal/mol (1650-fold). The N119D mutation also weakened 8-oxo-dGMP binding but only by 2.1 kcal/mol (37-fold), suggesting that Asn-119 functioned both as a hydrogen bond donor to C8O, and a hydrogen bond acceptor from N7H of 8-oxo-dGMP, while aspartate at position −119 functioned as an acceptor of a single hydrogen bond. Much smaller weakening effects (0.3–0.4 kcal/mol) on the binding of dGMP and dAMP were found, indicating specific hydrogen bonding of Asn-119 to 8-oxo-dGMP. While formation of the wild type MutT–Mg 2+-8-oxo-dGMP complex slowed the backbone NH exchange rates of 45 residues distributed throughout the protein, the same complex of the N119A mutant slowed the exchange rates of only 11 residues at or near the active site, indicating an increase in conformational flexibility of the N119A mutant. The R78K and R78A mutations weakened the binding of 8-oxo-dGMP by 1.7 and 1.1 kcal/mol, respectively, indicating a lesser role of Arg-78 than of Asn-119 in the selective binding of 8-oxo-dGMP, likely donating a single hydrogen bond to its C6O. The R78K+N119A double mutant weakened the binding of 8-oxo-dGMP ( K I slope=3.1 mM) by 6.5±0.2 kcal/mol which overlaps, within error with the sum of the effects of the two single mutants (6.0±0.3 kcal/mol). Such additive effects of the two single mutants in the double mutant are most simply explained by the independent functioning of Asn-119 and Arg-78 in the binding of 8-oxo-dGMP. Independent functioning of these two residues in nucleotide binding is consistent with their locations in the MutT–Mg 2+-8-oxo-dGMP complex, on opposite sides of the active site cleft, with a distance of 8.4±0.5 Å between their side chain nitrogens.