1 Mechanistic Principles of Enzyme-catalyzed Cleavage of Phosphodiester Bonds

I. INTRODUCTION Phosphodiester bonds, such as those found in both DNA and RNA, are extraordinarily resistant to hydrolysis. For example, the halftime for hydrolysis of the simplest phosphodiester, dimethyl phosphate, in 1 M NaOH is approximately 15 years at 35°C (Chin et al. 1989). Although this stability is important for maintenance of genomic integrity, it poses a challenging problem to enzymes involved in the synthesis, repair, and degradation of both DNA and RNA. If reactions involving the phosphodiester backbones of nucleic acids are to occur rapidly in living organisms, the catalytic power of these enzymes must be formidable. In fact, hydrolysis of the phosphodiester bonds in DNA catalyzed by staphylococcal nuclease is accelerated by a factor of ≥10 16 relative to the rate of the spontaneous reaction in the absence of enzyme (Serpersu et al. 1987). Although large, this rate acceleration is typical of protein enzymes that catalyze both hydrolysis and transesterification reactions of nucleic acids. The impressive catalytic power of these enzymes demands both mechanistic analysis and quantitative description. If the rate accelerations characteristic of enzyme-catalyzed phosphodiester bond hydrolysis could be understood quantitatively, the principles so elucidated could aid in the design of synthetic catalysts for (site-specific?) DNA and RNA degradation. The extreme resistance of phosphodiester bonds to nucleophilic attack (as evidenced by their extremely slow hydrolysis in concentrated base) can be attributed, in part, to electrostatic repulsion between the phosphodiester anion and the approaching nucleophile, either neutral water or hydroxide anion (Westheimer 1987). This simple consideration is not, however,...