NMR Evidence for an Unusually Low N1 pKa for Uracil Bound to Uracil DNA Glycosylase: Implications for Catalysis

Published

March 1, 2000

Author(s)

A C. Drohat, J T. Stivers

Abstract

The first step of the DNA base-excision repair pathway is the removal of the damaged base by cleavage of the N-glycosidic bond through the action of a DNA glycosylase. A key mechanistic question in these reactions is how the enzyme activates the expulsion of the leaving base. Solution and enzymatic studies have shown that purine bases may be activated by protonation or alkylation at N1, N3, or N7. However, activation of a pyrimidine base by a similar mechanism involving protonation at O2 or O4 is energetically untenable due to the low pK_a (difference}-3.4) of these carbonyl oxygens. Thus, it is not obvious how pyrimidine specific DNA glycosylases activate the leaving group base. A useful system to study these questions in detail is the relatively small and structurally well-characterized enzyme, uracil DNA glycosylase (UDG). This enzyme uses base-flipping and a simple hydrolytic mechanism to cleave the glycosidic bond of deoxyuridine in duplex and single strand DNA, giving the products uracil and abasic DNA. We have recently reported NMR and kinetic evidence which indicates that UDG activates the uracil-leaving group by providing a short hydrogen bond from a neutral His187 to the developing negative charge on uracil O2 in the transition state. Recent ¹H NMR experiments in our lab have revealed a highly deshielded resonance (δ = 15.6 ppm) in the ternary product complex at neutral pH that has been unambiguously assigned to this hydrogen bond. This type of catalysis, involving a neutral imidazole and an enolate-like oxygen, is similar to that proposed for several enzymes that catalyze proton abstraction from a carbon atom alpha to a carbonyl group, and becomes energetically more favorable as the difference in pKa between the enolate and imidazole is decreased. We show here, using a novel heteronuclear NMR approach, that the bound uracil base is anionic and in the N1 - O2 imidate form at pH 7.5. These findings support our previously proposed mechamism that requires significant charge resonance to uracil O2, and are inconsistent with recent proposals suggesting that a cationic histidine is involved in catalysis and that the negative charge resonates predominantly to uracil O4.

Citation

Journal of the American Chemical Society

Volume

122

Issue

No. 8

Pub Type

Journals

Keywords

catalysis, E coli uracil DNA glycosylase, hydrogen bond, leaving group activation, nuclear magnetic resonance spectroscopy, uracil N1 pK2

Citation

Drohat, A. and Stivers, J. (2000), NMR Evidence for an Unusually Low N1 pK_a for Uracil Bound to Uracil DNA Glycosylase: Implications for Catalysis, Journal of the American Chemical Society (Accessed November 8, 2024)