Role of electrophilic and general base catalysis in the mechanism of Escherichia coli uracil DNA glycosylase

Escherichia coli uracil DNA glycosylase (UDG) catalyzes the hydrolysis of p remutagenic uracil bases in DNA by flipping the deoxyuridine from the DNA h elix [Stivers, J. T., et al. (1999) Biochemistry 38, 952]. A general acid-b ase mechanism has been proposed whereby His187 facilitates leaving group de parture by protonating the O2 of uracil and Asp64 activates a water molecul e for nucleophilic attack at Cl' of the deoxyribose. Detailed kinetic studi es on the H187Q, H187A, and D64N mutant enzymes indicate that Asp64 and His 187 stabilize the chemical transition state by 5.3 and 4.8 kcal/mol, respec tively, with little effect on substrate or product binding. The pH dependen ce of k(cat) for wild-type and H187Q UDG indicates that an unprotonated gro up in the enzyme-substrate complex (pK(a) = 6.2 +/- 0.2) is required for ca talysis. This unprotonated group has a small Delta H of ionization (-0.4 +/ - 1.7 kcal/mol) and is absent in the pH profile for D64N UDG, suggesting th at it corresponds to the general base Asp64. The pH dependence of k(cat) fo r wild-type, H187Q, and D64N UDG shows no evidence for an essential protona ted group over the pH range of 5.5-10. Hence, the pK(a) of His187 must be o utside this pH range if-it serves as an electrophilic catalyst. These resul ts support a mechanism in which Asp64 serves as the general base and His187 acts as a neutral electrophile, stabilizing a developing negative charge o n uracil O2 in the transition state. In the following paper of this issue w e establish by crystallography and heteronuclear NMR spectroscopy that the imidazole of His 187 is neutral during the catalytic cycle of UDG.