Powering DNA repair through substrate electrostatic interactions.
The reaction catalyzed by the DNA repair enzyme uracil DNA glycosylase (UDG) proceeds through an unprecedented stepwise mechanism involving a positively charged oxacarbenium ion sugar and uracil anion leaving group. Here we use a novel approach to evaluate the catalytic contribution of electrostatic interactions between four essential phosphodiester groups of the DNA substrate and the cationic transition state. Our strategy was to substitute each of these phosphate groups with an uncharged (R)- or (S)-methylphosphonate linkage (MeP). We then compared the damaging effects of these methylphosphonate substitutions on catalysis with their damaging effects on binding of a cationic 1-azadeoxyribose (1-aza-dR(+)) oxacarbenium ion analogue to the UDG-uracil anion binary complex. A plot of log k(cat)/K(m) for the series of MeP-substituted substrates against log K(D) for binding of the 1-aza-dR(+) inhibitors gives a linear correlation of unit slope, confirming that the electronic features of the transition state resemble that of the 1-aza-dR(+), and that the anionic backbone of DNA is used in transition state stabilization. We estimate that all of the combined phosphodiester interactions with the substrate contribute 6-8 kcal/mol toward lowering the activation barrier, a stabilization that is significant compared to the 16 kcal/mol catalytic power of UDG. However, unlike groups of the enzyme that selectively stabilize the charged transition state by an estimated 7 kcal/mol, these phosphodiester groups also interact strongly in the ground state. To our knowledge, these results provide the first experimental evidence for electrostatic stabilization of a charged enzymatic transition state and intermediate using the anionic backbone of DNA.
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
Song
Fenhong
F
Stivers
James T
JT
D002384:Catalysis; D045647:DNA Glycosylases; D004260:DNA Repair; D004789:Enzyme Activation; D004795:Enzyme Stability; D008956:Models, Chemical; D009699:N-Glycosyl Hydrolases / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010755:Organophosphates / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000737:chemistry; D055672:Static Electricity; D013237:Stereoisomerism; D013379:Substrate Specificity; D013873:Thionucleotides / Q000737:chemistry; D051981:Uracil-DNA Glycosidase
D009838:Oligodeoxyribonucleotides; D010755:Organophosphates; D009943:Organophosphorus Compounds; D013873:Thionucleotides; C032627:methylphosphonic acid; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase