Abstract
The DNA repair enzyme uracil DNA glycosylase (UDG) hydrolyzes the glycosidic bond of deoxyuridine in DNA by a remarkable mechanism involving formation of a positively charged oxacarbenium ion-uracil anion intermediate. We have proposed that the positively charged intermediate is stabilized by being sandwiched between the combined negative charges of the anionic uracil leaving group and a conserved aspartate residue that are located on opposite faces of the sugar ring. Here we establish that a duplex DNA oligonucleotide containing a cationic 1-aza-deoxyribose (I) oxacarbenium ion mimic is a potent inhibitor of UDG that binds tightly to the enzyme-uracil anion (EU(-)) product complex (K(D) of EU(-) = 110 pm). The tight binding of I to the EU(-) complex results from its extremely slow off rate (k(off) = 0.0008 s(-1)), which is 25,000-fold slower than substrate analogue DNA. Removal of Asp(64) and His(187), which are involved in stabilization of the cationic sugar and the anionic uracil leaving group, respectively, specifically weakens binding of I to the UDG-uracil complex by 154,000-fold, without significantly affecting substrate or product binding. These results suggest that electrostatic effects can effectively stabilize such an intermediate by at least -7 kcal/mol, without leading to anticatalytic stabilization of the substrate and products.
Highlights
A long held viewpoint of enzymatic catalysis is that enzymes must form differential interactions with the ground state and transition state conformations of the substrate such that the activation barrier is diminished as compared with the corresponding reaction in the absence of the enzyme [1]
Overall Experimental Design and Rationale—The primary purpose of the current studies is to evaluate the magnitude and mechanism by which Uracil DNA glycosylase (UDG) stabilizes the putative oxacarbenium ion-uracil anion intermediate that was implicated in recent kinetic isotope effect (KIE) studies [10]
Features of the active site that contributed to this surprising structure were the use of binding interactions with the 3Ј- and 5Ј-phosphodiester groups to enforce a flattened sugar pucker that maximizes stabilizing hyperconjugative effects in the transition state and electrostatic stabilization by Asp64 and the uracil anion leaving group
Summary
Nucleoside Phosphoramidite and Oligonucleotide Synthesis—The nucleoside phosphoramidites were purchased from Applied Biosystems or Glen Research (Sterling, VA), except for the  anomer of the pyrene nucleoside phosphoramidite, the 2Ј--fluoro-2Ј-deoxyuridine phosphoramidite, and the 1-aza-1,2-dideoxy-4␣-carba-D-ribitol phosphoramidite, which were synthesized as described [12,13,14]. Fluorescence Measurements of the Association and Dissociation Rate Constants—The observed rate constants for association of AIA/TYT with the UDG-uracil anion complex were obtained using an Applied Photophysics 720 stopped flow fluorescence instrument (Surray, UK) under pseudo-first-order conditions in which the concentration of the EUϪ complex was always more than 4-fold greater than the concentration of AIA/TYT In these experiments a syringe containing a solution of UDG (200 –1800 nM) and uracil (3 mM) was rapidly mixed with a solution of AIA/TYT (50 nM) delivered from a second syringe, and the fluorescence change as a function of time was recorded using a 360-nm cut-off filter with excitation at 333 nm. The uracil concentration was maintained at 3 mM so that the enzyme was nearly entirely in the EU form at all pH values [8]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
