Inhibition of uracil DNA glycosylase by an oxacarbenium ion mimic.
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.
Jiang
Yu Lin
YL
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205-2185, USA.
Ichikawa
Yoshitaka
Y
Stivers
James T
JT
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
D001372:Aza Compounds; D009841:Oligonucleotides; D014498:Uracil; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase