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

Ricin toxin A-chain (RTA) exerts its cytotoxicity by depurinating 28 S ribo somal RNA at ribonucleotide A4234, a site of eukaryotic elongation factor b inding. Small stem-loop RNAs and DNAs can also be depurinated at the first adenine residue of 5'-GAGA-3' tetraloops. DNA oligonucleotides were synthes ized with DNA polymerase, using dATP labeled with [1'-C-14], [5'-C-14,9-N-1 5], [1'-H-3], [2'R-H-3], [2'S-H-3] or [5'-H-3]. Kinetic isotope effects (KI Es) were measured for depurination of a stem-loop DNA, called dA-10, with t he sequence d(5'-GGCGAGAGCC-3'). The commitment to catalysis was measured a nd found to be negligible, indicating that the experimental KIEs are the in trinsic KIEs of the chemical steps. The experimental KIEs, especially the s mall primary 1'-C-14 KIE of 1.015 +/- 0.001, demonstrated that the reaction proceeds through a stepwise D-N*A(N) mechanism, forming a discrete oxocarb enium.RTA complex with a lifetime greater than or equal to 10(-12) s. The s econdary KIEs at 1'-H-3, 2'R-H-3, and 2'S-H-3 were large and normal, suppor ting the existence of an oxocarbenium ion. In a stepwise mechanism, the KIE s are a function of the partitioning of the oxocarbenium ion intermediate f orward to products or back to reactants. KIEs calculated from density funct ional theory (DFT) optimized structures to quantitate partitioning of the i ntermediate were similar to each other and could not be used to determine p artitioning from the experimental KIEs. The large 2'R-3H and 2'S-3H KIEs de monstrate that the ribosyl ring in the oxocarbenium ion adopts a conformati on 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 hypercon jugation (Chen, X.-Y.; Berti, P. J.; Schramm, V. L. J. Am. Chem. Sec. 2000, 122, 1609-1617). The transition state for RNA hydrolysis by RTA is dominat ed by stem-loop geometry while that for DNA permits hyperconjugative stabil ization to determine the transition-state geometry.