The Role of Hydrogen Bonding in Transition-State Stabilization by Uracil-DNA Glycosylase

Alex Drohat Center for Advanced Research in Biotechnology (CARB)

An important question regarding the mechanism of N-glycosidic bond cleavage by pyrimidine-specific DNA glycosylases is how the enzyme activates the pyrimidine leaving group for expulsion. Recent studies in our lab indicate that Escherichia coli uracil DNA glycosylase (UDG) provides a hydrogen bond from a neutral His187 to stabilize the developing negative charge on uracil O2 in the transition state by 20 kJ/mol (Drohat, A.C. et al. Biochemistry 1999, 38, 11876-86). We show here that uracil bound to the product complex at neutral pH is in the N1-O2 imidate form and has an N1 pKa = 6.4 ± 0.1. This pKa is a surprising 3.4 units lower than for free uracil, corresponding to 20 kJ/mol of stabilization energy by the enzyme. Thus, the negative charge that develops on the uracil base during glycosidic bond cleavage resonates to O2 and not O4. This is consistent with the observation of a highly deshielded 1H NMR resonance (d = 15.6 ppm) that is assigned to a hydrogen bond from His187-Ne 2 to uracil O2. The D/H fractionation factor (f = 1.0 ± 0.1), solvent exchange rate and protection factor (kex = 7 s-1 and P.F. = 500), and change in 15N chemical shift upon hydrogen bond formation (D d = 10 ppm) indicate that this is a fairly strong, short hydrogen bond. These findings suggest a general mechanism for activation of pyrimidine leaving groups by DNA glycosylases involving a preorganized active site that has been highly evolved to solvate the developing negative charge in a concerted transition state as nucleophilic attack at C1¢ proceeds.