Role of Electrophilic and General Base Catalysis in the Mechanism of Escherichia coli Uracil DNA Glycosylase

Abstract

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 DeltaH 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.

Turning On Uracil-DNA Glycosylase Using a Pyrene Nucleotide Switch

“…Thus, the null effect of pyrene substitution for this mutant provides a dramatic demonstration of the specificity of the pyrene rescue for mutations that affect the base-flipping step. A similar null pyrene effect was obtained with the H187G mutant (not shown), which forms a strong interaction with uracil O 2 in the transition state, yet also has little effect on DNA binding or base flipping (17). We anticipate that pyrene rescue will be a useful tool to further elucidate the functional roles of other participants in base flipping such as the serine side chains involved in phosphodiester compression (pinching).…”

“…Comparison of the k cat values for the duplex substrates containing U/A, U/P, and U/Y pairs, shows that pyrene enhances k cat by 3.5-to 8-fold, which is similar to the enhancement seen with ssU single-stranded DNA (2.4-to 5.6-fold). We have previously shown in single turnover experiments that uracil is excised from single-stranded and duplex DNA forms with equal facility by UDG (ϳ120 s Ϫ1 ) and that the enhanced reactivity of ssDNA under k cat conditions is due to its faster dissociation from the product complex (8,17). The same phenomenon likely explains the increased k cat of the substrate with the U/Y pair observed here.…”

“…Purification of UDG-As previously described, UDG from E. coli strain B was purified to Ͼ99% homogeneity using a T7 polymerasebased overexpression system (9,17). The concentration of the enzyme was determined using an extinction coefficient of 38.511 mM Ϫ1 cm Ϫ1 (18).…”

“…In addition, the pyrene rescue hypothesis predicts that other mutations of UDG that do not affect the baseflipping step would not be rescued by pyrene substitution. Two such mutations that have been previously investigated by our group are D64N, which removes the water-activating group, and H187G, which removes the catalytic electrophile (17,24,25). Thus, these mutant enzymes were also tested with the pyrene substrates.…”

“…Catalytic Activity and DNA Binding of wtUDG with U/A, U/P, and U/Y Duplexes-The steady-state and single-turnover kinetic activity of wtUDG has been extensively studied in this laboratory using a variety of duplex and single-stranded substrate DNA molecules (5,17). This work has led to the conclusion that the overall rate for UDG under k cat conditions is severely limited by the product release step.…”