Transition State Analysis of Acid-Catalyzed dAMP Hydrolysis

Abstract

Multiple kinetic isotope effects (KIEs) on deoxyadenosine monophosphate (dAMP) hydrolysis in 0.1 M HCl were used to determine the transition state (TS) structure and probe its intrinsic reactivity. The experimental KIEs revealed a stepwise (SN1) mechanism, with a discrete oxacarbenium ion intermediate. This is the first direct evidence for the deoxyribosyl oxacarbenium ion in solution. In 50% methanol/0.1 M HCl the products were deoxyribose 5-phosphate (dRMP) and alpha- and beta-methyl dRMP. The alpha-Me-dRMP/beta-Me-dRMP ratio was 8.5:1. Assuming that a free oxacarbenium ion is equally susceptible to nucleophilic attack on either face, this indicated that approximately 20% proceeded through a solvent-separated ion pair complex, or free oxacarbenium ion, a DN+AN mechanism, while approximately 80% of the reaction proceeded through a contact ion pair complex. The oxacarbenium ion lifetime was estimated at 10(-11)-10(-10) s. Computational transition states were found for ANDN, DN*AN, DN*AN, and DN+AN mechanisms using hybrid density functional theory calculations. After taking into account 20% of DN+AN, there was an excellent match of calculated to experimental KIEs for 80% of the reaction having a DN*AN mechanism. That is, C-N bond cleavage is reversible, with dAMP and the {oxacarbenium ion*adenine} complex in equilibrium. The first irreversible step is water attack on the oxacarbenium ion. The calculated 1'-14C KIE for a stepwise mechanism with irreversible C-N bond cleavage (DN*AN) was 1.052, in the range previously associated only with ANDN transition states, and close to the calculated ANDN value, 1.059. The 1'-14C KIE was strongly dependent on the adenine protonation state.

Mechanisms for enzymatic cleavage of the N-glycosidic bond in DNA

“…23–26 As indicted in Fig. 2, these reactions are nearly always irreversible, though one DNA glycosylase can catalyze the synthesis of an N -glycosidic bond, using abasic DNA and one particular nucleobase.…”

“…23 The KIEs and other findings indicate that the first step, C-N bond cleavage, is rate-limiting, that is, a D N ‡ *A N mechanism. 23, 26, 31 …”

Potentially Prebiotic Synthesis of Pyrimidine β‐D‐Ribonucleotides by Photoanomerization/Hydrolysis of α‐D‐Cytidine‐2′‐Phosphate

“…Acid-catalyzed hydrolysis of b-d-uridine in the absence of irradiation proceeds through the related iminium ion 13 [11] by what is presumably a dissociative concerted process. [12] By analogy, we assume that the photochemically-generated species 11 and 12 also undergo hydrolysis by S N 2-type reactions, but at pH values near to neutrality.…”

Role of Base Excision “Repair” Enzymes in Erasing Epigenetic Marks from DNA

“…32,82,88 Protonation of dUrd is highly unfavorable, with a p K a of about −3.4 at O4 (Figure 9C). 80 As such, acid catalysis of dUrd hydrolysis is ineffective at neutral pH, but is observed as the pH approaches the p K a .…”