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 valenc es. In general, oxyphosphoranes with more negative charges are less st able. Although dianionic oxyphosphoranes do not have significant lifet imes, at least in the gas phase, a protonation of the dianionic specie s will enhance its stability. It is of particular interest that the pr eferred location for protonation of the monoanionic intermediate is fo und to be in the region between axial and equatorial oxygens despite t he fact that most of the negative charges are localized on equatorial oxygens. Placement of the proton between the most negatively charged e quatorial oxygens leads to a transition state with a rotation of the P -O-equatorial(H) bond. Further, we examined the kinetic stability of t he dianionic phosphorane neutralized either by two protons or by a div alent magnesium ion. Neutralization by two protons increases the stabi lity of the resulting phosphorane. On the other hand, unexpectedly, th e neutral complex between the dianionic phosphorane and the divalent m agnesium ion does not have a lifetime. Moreover, the location of the m agnesium ion at the frozen configuration of the pentacoordinate interm ediate is also found to be in the region between the axial and equator ial oxygens. When all the frozen parameters are completely relaxed, th e oxyphosphorane undergoes decomposition by breaking a phosphorus-oxyg en bond. These results support the idea that ribozymes are metalloenzy mes and magnesium ion itself is capable of cleaving (or forming from t he principle of microscopic reversibility) of a phosphorus-oxygen bond by a direct coordination to the translating oxygen. Kinetic data on s ynthetic ribozymes are in agreement with this interpretation.