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

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• 作者：Vibhor Saraswat ; Hugo F. Azurmendi ; and Albert S. Mildvan
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：March 30, 2004
• 年：2004
• 卷：43
• 期：12
• 页码：3404 - 3414
• 全文大小：441K
• 年卷期：v.43,no.12(March 30, 2004)
• ISSN：1520-4995

文摘

The solution structure of the ternary MutT enzyme-Mg²⁺-8-oxo-dGMP complex showedthe proximity of Asn119 and Arg78 and the modified purine ring of 8-oxo-dGMP, suggesting specificroles 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 rolesare 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 (s/entities/le.gif">2-fold) and K_m (s/entities/le.gif">4-fold) in the hydrolysis ofdGTP, indicating largely intact active sites. ¹H-¹⁵N HSQC spectra showed largely intact protein structuresfor all of these mutants. However, the N119A mutation profoundly and selectively weakened the activesite binding of 8-oxo-dGMP, increasing the K_I^slope of this product inhibitor 1650-fold, while increasingthe 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=Ogroup and accepted a hydrogen bond from the N7H group of 8-oxo-dGMP, while Asp119 functioned asonly an acceptor. Direct binding of 8-oxo-dGMP to N119A, monitored by continuous changes in the ¹⁵Nand/or NH chemical shifts of 12 residues, revealed fast exchange, and a K_D of 237 ± 130 s/entities/mgr.gif">M for 8-oxo-dGMP, comparable to its K_I^slope of 81 ± 22 s/entities/mgr.gif">M. 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 theactive site, indicating an increase in the conformational flexibility of the N119A mutant. The R78K andR78A 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 ahydrogen bond to its C6=O group. The much greater contribution of Asn119 (4.0 kcal/mol) than ofArg78 (1.0 kcal/mol) to the selectivity of binding of 8-oxo-dGMP versus dGMP indicates a 2 order ofmagnitude smaller contribution of a structure with the reversed orientation of the 8-oxo-dG ring. TheR78K/N119A double mutant weakened the binding of 8-oxo-dGMP by a factor (63 000 ± 22 000) whichoverlaps within error with the product of the effects of the two single mutants (28 000 ± 15 000). Suchadditive effects of the two single mutants in the double mutant are most simply explained by the independentfunctioning of Asn119 and Arg78 in the binding of 8-oxo-dGMP. Independent functioning of these tworesidues in nucleotide binding is consistent with their locations in the MutT-Mg²⁺-8-oxo-dGMP complex,on opposite sides of the active site cleft, with a minimal distance of 8.4 ± 0.5 Å between their side chainnitrogens.