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 P
, 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
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 toelucidate the kinetic scheme, product inhibition by 8-oxo-dGMP
and dGMP
and direct binding of thesenucleotides to MutT were studied. With Mg
2+-activated dGTP hydrolysis, 8-oxo-dGMP is a noncompetitiveinhibitor with
KIslope = 49 nM, which is 10
4.6-fold lower than the
KIslopeof dGMP (1.7 mM). Similarly, the
KIintercept 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
KD = 1.8 mM, in agreement with its
KIslope. With 8-oxo-dGMP, tightbinding
and slow exchange (
n = 1.0 ± 0.1,
KD < 0.25 mM) are found. Isothermal calorimetric titrationof MutT with 8-oxo-dGMP yields a
KD of 52 nM, in agreement with its
KIslope. Changing the metal activatorfrom Mg
2+ to Mn
2+ had little effect on the
KIslope of dGMP or of 8-oxo-dGMP, consistent with the second-sphere enzyme-M
2+-H
2O-NTP-M
2+ 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
KIof 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 (
G = -9.8 kcal/mol) is driven by a highly favorable enthalpy(<
Hbinding> = -32 ± 7 kcal/mol), with an unfavorable entropy (<-
> = +22 ± 7 kcal/mol), asdetermined by van't Hoff analysis of the effect of temperature on the
KIslope and by isothermal titrationcalorimetry 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 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.