Phosphodiester linkages, including those that join the nucleotides of DNA, are highly resistant to spontaneous hydrolysis. The rate of water attack at the phosphorus atom of phosphodiesters is known only as an upper limit, based on the hydrolysis of the dimethyl phosphate anion. That reaction was found to proceed at least 99% by C–O cleavage, at a rate suggesting an upper limit of 10 ⁻¹⁵ s ⁻¹ for P–O cleavage of phosphodiester anions at 25°C. To evaluate the rate enhancement produced by P–O cleaving phosphodiesterases such as staphylococcal nuclease, we decided to establish the actual value of the rate constant for P–O cleavage of a simple phosphodiester anion. In dineopentyl phosphate, C–O cleavage is sterically precluded so that hydrolysis occurs only by P–O cleavage. Measurements at elevated temperatures indicate that the dineopentyl phosphate anion undergoes hydrolysis in water with a t _1/2 of 30,000,000 years at 25°C, furnishing an indication of the resistance of the internucleotide linkages of DNA to water attack at phosphorus. These results imply that staphylococcal nuclease ( k _cat = 95 s ⁻¹ ) enhances the rate of phosphodiester hydrolysis by a factor of ≈10 ¹⁷ . In alkaline solution, thymidylyl-3′-5′-thymidine (TpT) has been reported to decompose 10 ⁵ -fold more rapidly than does dineopentyl phosphate. We find however that TpT and thymidine decompose at similar rates and with similar activation parameters, to a similar set of products, at pH 7 and in 1 M KOH. We infer that the decomposition of TpT is initiated by the breakdown of thymidine, not by phosphodiester hydrolysis.

To evaluate the proficiency of phosphatases as catalysts, the rate of the uncatalyzed hydrolysis of simple phosphate monoester dianions was estimated by extrapolating rates measured over a range of high temperatures. The rate of spontaneous hydrolysis of phenyl phosphate dianion indicates that a linear free energy relationship reported earlier is reliable for leaving groups whose conjugate acids have pKa values up to at least 10. Using Teflon reaction vessels, it proved possible to follow the hydrolysis of methyl phosphate and 3-(4-carboxy)-2,2-dimethylpropyl phosphate in strong alkali. Even in 1 M KOH, the reaction was found to be specific acid catalyzed. These results establish an upper limit for dianion reactivity, which had been overestimated earlier as a result of the leaching by alkali of silicic acid from quartz reaction vessels. The present findings indicate that the half-time for attack by water on alkyl phosphate dianions is 1.1 × 10 ¹² years ( k = 2 × 10 ⁻²⁰ s) at 25°C and that phosphatases involved in cell signaling and regulation produce the largest rate enhancements that have been identified thus far. Protein phosphatase-1 and inositol 1-phosphatase exceed all other known enzymes in their affinities for the altered substrates in the transition state.