Escherichia coli uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil bases in DNA by flipping the deoxyuridine from the DNA helix Stivers, J. T., et al. (1999) Biochemistry 38, 952. A general acid−base mechanism has been proposed whereby His187 facilitates leaving group departure by protonating the O2 of uracil and Asp64 activates a water molecule for nucleophilic attack at C1‘ of the deoxyribose. Detailed kinetic studies on the H187Q, H187A, and D64N mutant enzymes indicate that Asp64 and His187 stabilize the chemical transition state by 5.3 and 4.8 kcal/mol, respectively, with little effect on substrate or product binding. The pH dependence of k cat for wild-type and H187Q UDG indicates that an unprotonated group in the enzyme−substrate complex (pK a = 6.2 ± 0.2) is required for catalysis. This unprotonated group has a small ΔH of ionization (−0.4 ± 1.7 kcal/mol) and is absent in the pH profile for D64N UDG, suggesting that it corresponds to the general base Asp64. The pH dependence of k cat for wild-type, H187Q, and D64N UDG shows no evidence for an essential protonated group over the pH range of 5.5−10. Hence, the pK a of His187 must be outside this pH range if it serves as an electrophilic catalyst. These results support a mechanism in which Asp64 serves as the general base and His187 acts as a neutral electrophile, stabilizing a developing negative charge on uracil O2 in the transition state. In the following paper of this issue we establish by crystallography and heteronuclear NMR spectroscopy that the imidazole of His187 is neutral during the catalytic cycle of UDG.