Evidence for direct attack by hydroxide in phosphodiester hydrolysis

Area of Science:

• Biochemistry
• Chemical Kinetics

Background:

• Phosphodiester hydrolysis is crucial in biological processes.
• The role of nucleophiles in these reactions remains incompletely understood.

Purpose of the Study:

• To elucidate the mechanism of hydroxide in thymidine-5'-p-nitrophenyl phosphate hydrolysis.
• To determine if hydroxide acts as a nucleophile or a general base.

Main Methods:

• Measured solvent deuterium isotope effects (D2Ok).
• Analyzed ionic strength effects.
• Determined 18O isotope effects on the solvent nucleophile (18knuc).

Main Results:

• Slightly inverse D2Ok suggests no proton transfer in the transition state.
• Hydroperoxide showed a significant alpha effect, indicating nucleophilic attack by oxyanions.
• Indistinguishable ionic strength dependencies for hydroxide and hydroperoxide suggest a shared mechanism.
• 18knuc exceeded the equilibrium 18O isotope effect, supporting direct nucleophilic attack by hydroxide.

Conclusions:

• Data strongly support direct nucleophilic attack by hydroxide in phosphodiester hydrolysis.
• A large kinetic component of the 18O isotope effect indicates minimal bond order formation in the transition state.