Mutational, NMR, and NH Exchange Studies of the Tight and Selective Binding of 8-Oxo-dGMP by the MutT Pyrophosphohydrolase

• Published in: Biochemistry (Easton) 2004-03, Vol.43 (12), p.3404-3414
• Main Authors: Saraswat, Vibhor, Azurmendi, Hugo F, Mildvan, Albert S
• Format: Article
• Language: English
• Subjects: Alanine - genetics; Arginine - genetics; Asparagine - genetics; Binding Sites - genetics; Deuterium Exchange Measurement; DNA Mutational Analysis; Escherichia coli Proteins - antagonists & inhibitors; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Guanosine Monophosphate - analogs & derivatives; Guanosine Monophosphate - chemistry; Hydrogen Bonding; Kinetics; Ligands; Magnesium - chemistry; Mutagenesis, Site-Directed; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular; Phosphoric Monoester Hydrolases - antagonists & inhibitors; Phosphoric Monoester Hydrolases - chemistry; Phosphoric Monoester Hydrolases - genetics; Protein Binding - genetics; Protons; Pyrophosphatases; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi030216o

The solution structure of the ternary MutT enzyme−Mg2+−8-oxo-dGMP complex showed the proximity of Asn119 and Arg78 and the modified purine ring of 8-oxo-dGMP, suggesting specific roles for these residues in the tight and selective binding of this nucleotide product [Massiah, M. A., Saraswat, V., Azurmendi, H. F., and Mildvan, A. S. (2003) Biochemistry 42, 10140−10154]. These roles are here tested by mutagenesis. The N119A, N119D, R78K, and R78A single mutations and the R78K/N119A double mutant showed very small effects on k cat (≤2-fold) and K m (≤4-fold) in the hydrolysis of dGTP, indicating largely intact active sites. 1H−15N HSQC spectra showed largely intact protein structures for all of these mutants. However, the N119A mutation profoundly and selectively weakened the active site binding of 8-oxo-dGMP, increasing the K I slope of this product inhibitor 1650-fold, while increasing the K I slope of dGMP and dAMP less than 2-fold. The N119D mutation also selectively weakened 8-oxo-dGMP binding but only by 37-fold, suggesting that Asn119 both donated a hydrogen bond to the C8O group and accepted a hydrogen bond from the N7H group of 8-oxo-dGMP, while Asp119 functioned as only an acceptor. Direct binding of 8-oxo-dGMP to N119A, monitored by continuous changes in the 15N and/or NH chemical shifts of 12 residues, revealed fast exchange, and a K D of 237 ± 130 μM for 8-oxo-dGMP, comparable to its K I slope of 81 ± 22 μM. While formation of the wild-type MutT−Mg2+−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 the conformational flexibility of the N119A mutant. The R78K and R78A mutations selectively increased the K I slope of 8-oxo-dGMP by factors of 17 and 6.6, respectively, indicating a smaller role for Arg78 than for Asn119 in the binding of 8-oxo-dGMP, likely donating a hydrogen bond to its C6O group. The much greater contribution of Asn119 (4.0 kcal/mol) than of Arg78 (1.0 kcal/mol) to the selectivity of binding of 8-oxo-dGMP versus dGMP indicates a 2 order of magnitude smaller contribution of a structure with the reversed orientation of the 8-oxo-dG ring. The R78K/N119A double mutant weakened the binding of 8-oxo-dGMP by a factor (63 000 ± 22 000) which overlaps within error with the product of the effects of the two single mutants (28 000 ± 15 000). Such a