Escherichia coli Uracil DNA Glycosylase:  NMR Characterization of the Short Hydrogen Bond from His187 to Uracil O2

Abstract

Uracil DNA glycosylase (UDG) cleaves the glycosidic bond of deoxyuridine in DNA using a hydrolytic mechanism, with an overall catalytic rate enhancement of 10(12)-fold over the solution reaction. The nature of the enzyme-substrate interactions that lead to this large rate enhancement are key to understanding enzymatic DNA repair. Using (1)H and heteronuclear NMR spectroscopy, we have characterized one such interaction in the ternary product complex of Escherichia coli UDG, the short (2.7 A) H bond between His187 N(epsilon)(2) and uracil O2. The H bond proton is highly deshielded at 15.6 ppm, indicating a short N-O distance and exhibits a solvent exchange rate that is 400- and 10(5)-fold slower than free imidazole at pH 7.5 and pH 10, respectively. Heteronuclear NMR experiments at neutral pH show that this H bond involves the neutral imidazole form of His187 and the N1-O2 imidate form of uracil. The excellent correspondence of the pK(a) for the disappearance of the H bond (pK(a) = 6.3 +/- 0.1) with the previously determined pK(a) = 6.4 for the N1 proton of enzyme-bound uracil indicates that the H bond requires negative charge on uracil O2 [Drohat, A. C., and Stivers, J. T. (2000) J. Am. Chem. Soc. 122, 1840-1841]. Although the above characteristics suggest a short strong H bond, the D/H fractionation factor of phi = 1.0 is more typical of a normal H bond. This unexpected observation may reflect a large donor-acceptor pK(a) mismatch or the net result of two opposing effects on vibrational frequencies: decreased N-H bond stretching frequencies (phi < 1) and increased bending frequencies (phi > 1) relative to the O-H bonds of water. The role of this H bond in catalysis by UDG and several approaches to quantify the H bond energy are discussed.

The acidity of uracil and uracil analogs in the gas phase: Four surprisingly acidic sites and biological implications

“…Therefore, in a nonpolar cavity, N1-deprotonated uracil may be easier to cleave than the solution phase pK a s imply. In keeping with our predictions, recent experimental studies with UDGase indicate that the anionic base is most probably a leaving group, contrary to previous hypotheses that involved protonation of the uracil before cleavage [22,[31][32][33].…”

Specificity of Human Thymine DNA Glycosylase Depends on N-Glycosidic Bond Stability

“…77,[80][81][82] For UDG, two conserved residues each contribute about 5 kcal/mol of TS stabilization; an Asp side-chain serves to stabilize the oxacarbenium cation and activate (or position) the water nucleophile, and a His side-chain stabilizes the uracil anion leaving group via formation of a strong H bond. 80,[83][84][85] Although these catalytic groups are absent in hTDG, the enzyme still provides over 12 kcal/mol of TS stabilization for a dU substrate (Table 2) as compared to 17 kcal/mol for UDG. 83 Structural studies of eMUG suggest that it and hTDG may provide some stabilization to an anionic nucleobase leaving group via H bond interactions involving backbone amides and an activesite water molecule.…”

Probing the Limits of Electrostatic Catalysis by Uracil DNA Glycosylase Using Transition State Mimicry and Mutagenesis

“…On the basis of this body of work, it was proposed that UDG stabilized the intermediate using an "electrostatic sandwich" mechanism involving a conserved aspartate residue and the negative charge provided by the uracil anion leaving group. Another key player in this proposed mechanism is the active site residue, His 187 , which has been shown to form a strong hydrogen bond to uracil O-2 and thereby stabilizes the N-1-O-2 enolate by 5 kcal/mol in the ternary product complex as compared with solution (8). The energetic role of the His 187 hydrogen bond is likely to be of comparable importance in stabilizing the transition state and the subsequent ionic intermediate.…”