Dataset Information

Ground State Destabilization in Uracil DNA Glycosylase: Let's Not Forget "Tautomeric Strain" in Substrates.

ABSTRACT: Enzymes like uracil DNA glycosylase (UDG) can achieve ground state destabilization, by polarizing substrates to mimic rare tautomers. On the basis of computed nucleus independent chemical shifts, NICS(1)zz, and harmonic oscillator model of electron delocalization (HOMED) analyses, of quantum mechanics (QM) and quantum mechanics/molecular mechanics (QM/MM) models of the UDG active site, uracil is strongly polarized when bound to UDG and resembles a tautomer >12 kcal/mol higher in energy. Natural resonance theory (NRT) analyses identified a dominant O2 imidate resonance form for residue bound 1-methyl-uracil. This "tautomeric strain" raises the energy of uracil, making uracilate a better than expected leaving group. Computed gas-phase SN2 reactions of free and hydrogen bonded 1-methyl-uracil demonstrate the relationship between the degree of polarization in uracil and the leaving group ability of uracilate.

SUBMITTER: Das R

PROVIDER: S-EPMC6726543 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Publications

Ground State Destabilization in Uracil DNA Glycosylase: Let's Not Forget "Tautomeric Strain" in Substrates.

Das Ranjita R Vázquez-Montelongo Erik A EA Cisneros G Andrés GA Wu Judy I JI

Journal of the American Chemical Society 20190826 35

Enzymes like uracil DNA glycosylase (UDG) can achieve ground state destabilization, by polarizing substrates to mimic rare tautomers. On the basis of computed nucleus independent chemical shifts, NICS(1)<sub><i>zz</i></sub>, and harmonic oscillator model of electron delocalization (HOMED) analyses, of quantum mechanics (QM) and quantum mechanics/molecular mechanics (QM/MM) models of the UDG active site, uracil is strongly polarized when bound to UDG and resembles a tautomer >12 kcal/mol higher i ...[more]

PMID: 31434485

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Project description:Two dimensional (2D) NMR and molecular dynamics simulations have been used to determine the three dimensional (3D) structure of a hairpin DNA, d-CTA-GAGGATCC-TUTT-GGATCCT (22mer; abbreviated as U2-hairpin), which has uracil at the second position from the 5' end of the tetraloop. The(1)H resonances of this hairpin have been assigned almost completely. NMR restrained molecular dynamics and energy minimization procedures have been used to describe the 3D structure of U2-hairpin. This study establishes that the stem of the hairpin adopts a right-handed B-DNA conformation, while the T(12)and T(15)nucleotides stack upon 3' and 5' ends of the stem, respectively. Further, T(14)stacks upon both T(12)and T(15). Though U(13)partially stacks upon T(14), no stacking interaction is observed between U(13)and T(12). All the individual nucleotide bases belonging to the stem and T(12)and T(15)of the loop adopt ' anti ' conformation with respect to their sugar moiety, while the U(13)and T(14)of the loop are in ' syn ' conformation. The turning phosphate in the loop is located between T(13)and T(14). This study and a concurrent NMR structural study on yet another hairpin DNA d-CTAGAGGAATAA-TTTU-GGATCCT (22mer; abbreviated as U4-hairpin), with uracil at the fourth position from the 5' end of the tetraloop throw light upon various interactions which have been reported between Escherichia coli uracil DNA glycosylase (UDG) and uracil containing DNA. The epsilon of T(12)and alpha, beta, gamma, epsilon and zeta of U(13)and gamma of T(14), which partially influence the local conformation of U(13)in U2-hairpin are all locked in ' trans ' conformation. Such stretched out backbone conformation in the vicinity of U(13)could be the reason as to why the U2-hairpin is found to be the poor substrate for its interaction with UDG compared to the other substrates in which the uracil is at first, third and fourth positions of the tetraloop from its 5' end, as reported earlier by Vinay and Varshney. This study shows that UDG actively promotes the flipping of uracil from a stacked conformation and rules out the possibility of UDG recognizing the flipped out uracil bases.

Project description:Enzymes stabilize transition states of reactions while limiting binding to ground states, as is generally required for any catalyst. Alkaline Phosphatase (AP) and other nonspecific phosphatases are some of Nature's most impressive catalysts, achieving preferential transition state over ground state stabilization of more than 10²²-fold while utilizing interactions with only the five atoms attached to the transferred phosphorus. We tested a model that AP achieves a portion of this preference by destabilizing ground state binding via charge repulsion between the anionic active site nucleophile, Ser102, and the negatively charged phosphate monoester substrate. Removal of the Ser102 alkoxide by mutation to glycine or alanine increases the observed Pi affinity by orders of magnitude at pH 8.0. To allow precise and quantitative comparisons, the ionic form of bound P(i) was determined from pH dependencies of the binding of Pi and tungstate, a P(i) analog lacking titratable protons over the pH range of 5-11, and from the ³¹P chemical shift of bound P(i). The results show that the Pi trianion binds with an exceptionally strong femtomolar affinity in the absence of Ser102, show that its binding is destabilized by ?10?-fold by the Ser102 alkoxide, and provide direct evidence for ground state destabilization. Comparisons of X-ray crystal structures of AP with and without Ser102 reveal the same active site and P(i) binding geometry upon removal of Ser102, suggesting that the destabilization does not result from a major structural rearrangement upon mutation of Ser102. Analogous Pi binding measurements with a protein tyrosine phosphatase suggest the generality of this ground state destabilization mechanism. Our results have uncovered an important contribution of anionic nucleophiles to phosphoryl transfer catalysis via ground state electrostatic destabilization and an enormous capacity of the AP active site for specific and strong recognition of the phosphoryl group in the transition state.

Dataset Information

Ground State Destabilization in Uracil DNA Glycosylase: Let's Not Forget "Tautomeric Strain" in Substrates.

Publications

Ground State Destabilization in Uracil DNA Glycosylase: Let's Not Forget "Tautomeric Strain" in Substrates.

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