Probing the limits of electrostatic catalysis by uracil DNA glycosylase using transition state mimicry and mutagenesis

Yu Lin Jiang, Alexander C. Drohat, Yoshitaka Ichikawa, James Stivers

Research output: Contribution to journalArticle

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 (KD of EU- = 110 pM). The tight binding of I to the EU- complex results from its extremely slow off rate (koff = 0.0008 s-1), which is 25,000-fold slower than substrate analogue DNA. Removal of Asp64 and His187, 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.

Original languageEnglish (US)
Pages (from-to)15385-15392
Number of pages8
JournalJournal of Biological Chemistry
Volume277
Issue number18
DOIs
Publication statusPublished - May 3 2002

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ASJC Scopus subject areas

  • Biochemistry

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