Transition-state analysis for depurination of DNA by Ricin A-chain

Ricin toxin A-chain (RTA) exerts its cytotoxicity by depurinating 28 S ribosomal RNA at ribonucleotide A4234, a site of eukaryotic elongation factor binding. Small stem-loop RNAs and DNAs can also be depurinated at the first adenine residue of 5′-GAGA-3′ tetraloops. DNA oligonucleotides were synthesized with DNA polymerase, using dATP labeled with [1′-¹⁴C], [5′-¹⁴C,9-¹⁵N], [1′-³H], [2′R-³H], [2′S-³H] or [5′-³H]. Kinetic isotope effects (KIEs) were measured for depurination of a stem-loop DNA, called dA-10, with the sequence d(5′-GGCGAGAGCC-3′). The commitment to catalysis was measured and found to be negligible, indicating that the experimental KIEs are the intrinsic KIEs of the chemical steps. The experimental KIEs, especially the small primary 1′-¹⁴C KIE of 1.015 ± 0.001, demonstrated that the reaction proceeds through a stepwise D_N* A_N mechanism, forming a discrete oxocarbenium·RTA complex with a lifetime ≥10^-12 s. The secondary KIEs at 1′-³H, 2′R-³H, and 2′S-³H were large and normal, supporting the existence of an oxocarbenium ion. In a stepwise mechanism, the KIEs are a function of the partitioning of the oxocarbenium ion intermediate forward to products or back to reactants. KIEs calculated from density functional theory (DFT) optimized structures to quantitate partitioning of the intermediate were similar to each other and could not be used to determine partitioning from the experimental KIEs. The large 2′R-³H and 2′S-³H KIEs demonstrate that the ribosyl ring in the oxocarbenium ion adopts a conformation giving maximal hyperconjugative stabilization of the cationic center at Cl′. This is in contrast to the RNA substrate where the nucleotide backbone constrains the ribosyl ring in a 3′-endo conformation with little hyperconjugation (Chen, X.-Y.; Berti, P. J.; Schramm, V. L. J. Am. Chem. Soc. 2000, 122, 1609-1617). The transition state for RNA hydrolysis by RTA is dominated by stem-loop geometry while that for DNA permits hyperconjugative stabilization to determine the transitionstate geometry.