title: Inhibition of uracil DNA glycosylase by an oxacarbenium ion mimic.
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 (K(D) = 2 microM) but binds 4000-fold more tightly to the enzyme-uracil anion (EU) product complex (K(D) = 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 microM(-1) x s(-1)). Accordingly, the low K(D) of AIA for the EU complex is largely due its very slow off-rate (5 x 10(-4) x 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.
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205-2185, USA.
authors.forename: Yu Lin
authors.initials: YL
authors.lastname: Jiang
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Ichikawa
authors.forename: James T
authors.initials: JT
authors.lastname: Stivers
mesh_terms: D001372:Aza Compounds / Q000494:pharmacology*; D045647:DNA Glycosylases; D004926:Escherichia coli; D018716:Molecular Mimicry; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D014498:Uracil / Q000378:metabolism*; D051981:Uracil-DNA Glycosidase
chemical_list: D001372:Aza Compounds; D009841:Oligonucleotides; D014498:Uracil; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase