THEORETICAL AND EXPERIMENTAL CONSIDERATIONS ON THE HAMMERHEAD RIBOZYME REACTIONS - DIVALENT MAGNESIUM-ION MEDIATED CLEAVAGE OF PHOSPHORUS-OXYGEN BONDS

Stabilities of oxyphosphoranes were examined with various ionic valences. In general, oxyphosphoranes with more negative charges are less stable. Although dianionic oxyphosphoranes do not have significant lifetimes, at least in the gas phase, a protonation of the dianionic species will enhance its stability. It is of particular interest that the preferred location for protonation of the monoanionic intermediate is found to be in the region between axial and equatorial oxygens despite the fact that most of the negative charges are localized on equatorial oxygens. Placement of the proton between the most negatively charged equatorial oxygens leads to a transition state with a rotation of the P-O-equatorial(H) bond. Further, we examined the kinetic stability of the dianionic phosphorane neutralized either by two protons or by a divalent magnesium ion. Neutralization by two protons increases the stability of the resulting phosphorane. On the other hand, unexpectedly, the neutral complex between the dianionic phosphorane and the divalent magnesium ion does not have a lifetime. Moreover, the location of the magnesium ion at the frozen configuration of the pentacoordinate intermediate is also found to be in the region between the axial and equatorial oxygens. When all the frozen parameters are completely relaxed, the oxyphosphorane undergoes decomposition by breaking a phosphorus-oxygen bond. These results support the idea that ribozymes are metalloenzymes and magnesium ion itself is capable of cleaving (or forming from the principle of microscopic reversibility) of a phosphorus-oxygen bond by a direct coordination to the translating oxygen. Kinetic data on synthetic ribozymes are in agreement with this interpretation.