Abstract
We have investigated the inhibition of the DNA repair enzyme uracil DNA glycosylase (UDG) by an 11-mer oligonucleotide (AIA) containing a cationic 1-aza-deoxyribose (I) residue designed to be a stable mimic of the high-energy oxacarbenium ion reaction intermediate [Werner, R. M., and Stivers, J. T. (2000) Biochemistry 39, 14054-14064]. Inhibition kinetics and direct binding studies indicate that AIA binds weakly to the free enzyme (KD = 2 μM) but binds 4000-fold more tightly to the enzymeuracil anion (EU) product complex (KD = 500 pM). The importance of the positive charge on the 1-nitrogen in binding is established by the observation that AIA binds <30 000-fold more tightly to the EU complex than the corresponding neutral tetrahydrofuran (F) abasic site product analogue (AFA). The unusual inhibition mechanism for AIA results in a time dependence that resembles slow-onset inhibition even though the apparent on-rate of the inhibitor for the EU- binary product complex is moderate (1 μM-1 s-1). Accordingly, the low KD of AIA for the EU complex is largely due its very slow off-rate (5 × 10-4 s-1). These results support previous kinetic isotope effect measurements that indicate UDG stabilizes a discrete oxacarbenium ion-uracil anion intermediate. This oxacarbenium ion mimic represents the tightest binding inhibitor of UDG yet identified.
Original language | English (US) |
---|---|
Pages (from-to) | 7116-7124 |
Number of pages | 9 |
Journal | Biochemistry |
Volume | 41 |
Issue number | 22 |
DOIs | |
State | Published - Jun 4 2002 |
ASJC Scopus subject areas
- Biochemistry