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

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• 作者：Vibhor Saraswat ; Michael A. Massiah ; Gregory Lopez ; L. Mario Amzel ; and Albert S. Mildvan
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：December 31, 2002
• 年：2002
• 卷：41
• 期：52
• 页码：15566 - 15577
• 全文大小：316K
• 年卷期：v.41,no.52(December 31, 2002)
• ISSN：1520-4995

文摘

The MutT enzyme from E. coli, in the presence of a divalent cation, catalyzes the hydrolysisof nucleoside- and deoxynucleoside-triphosphate (NTP) substrates by nucleophilic substitution at Ps/gifchars/beta2.gif" BORDER=0 ALIGN="middle">, toyield a nucleotide (NMP) and PPi. The best substrate of MutT is believed to be the mutagenic nucleotide8-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 toelucidate the kinetic scheme, product inhibition by 8-oxo-dGMP and dGMP and direct binding of thesenucleotides to MutT were studied. With Mg²⁺-activated dGTP hydrolysis, 8-oxo-dGMP is a noncompetitiveinhibitor with K_I^slope = 49 nM, which is 10^4.6-fold lower than the K_I^slopeof dGMP (1.7 mM). Similarly, theK_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. Noncompetitiveinhibition by dGMP and 8-oxo-dGMP indicates an "iso" mechanism in which the nucleotide productleaves an altered form of the enzyme which slowly reverts to the form which binds substrate. Consistentwith this kinetic scheme, ¹H-¹⁵N HSQC titration of MutT with dGMP reveals weak binding and fastexchange from one site with a K_D = 1.8 mM, in agreement with its K_I^slope. With 8-oxo-dGMP, tightbinding and slow exchange (n = 1.0 ± 0.1, K_D < 0.25 mM) are found. Isothermal calorimetric titrationof MutT with 8-oxo-dGMP yields a K_D of 52 nM, in agreement with its K_I^slope. Changing the metal activatorfrom Mg²⁺ to Mn²⁺ had little effect on the K_I^slope 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 K_Iof 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 (s/gifchars/Delta.gif" BORDER=0 >Gs/entities/deg.gif"> = -9.8 kcal/mol) is driven by a highly favorable enthalpy(<s/gifchars/Delta.gif" BORDER=0 >H_binding> = -32 ± 7 kcal/mol), with an unfavorable entropy (<-subscribe/journals/bichaw/41/i52/eqn/bi020552pe10001.gif">> = +22 ± 7 kcal/mol), asdetermined by van't Hoff analysis of the effect of temperature on the K_I^slope and by isothermal titrationcalorimetry in two buffer systems. The binding of 8-oxo-dGMP to MutT induces changes in backbone¹⁵N and NH chemical shifts of 62 residues widely distributed throughout the protein, while dGMP bindinginduces smaller changes in only 22 residues surrounding the nucleotide binding site, suggesting that theunusually high affinity of MutT for 8-oxo-nucleotides is due not only to interactions with the altered8-oxo or 7-NH positions on guanine, but results primarily from diffuse structural changes which tightenthe protein structure around the 8-oxo-nucleotide.