Phosphate Monoester Hydrolysis in Cyclohexane

Abstract

The hydrolysis of simple phosphate monoesters is among the most difficult reactions that are subject to catalysis by enzymes, and it has been suggested that extraction of the substrates from solvent water may contribute to the catalytic effects of phosphohydrolases. Here, we show that the tetrabutylammonium salt of neopentyl phosphate enters wet cyclohexane at concentrations sufficient to allow determination of its rate of hydrolysis. The second-order rate constant for hydrolysis of the phosphomonoester dianion is enhanced approximately 2 x 10(12)-fold by transfer from water to cyclohexane. That rate enhancement arises from an increase in the entropy of activation.

Enhancement of the Rate of Pyrophosphate Hydrolysis by Nonenzymatic Catalysts and by Inorganic Pyrophosphatase

“…The k cat value of 570 s Ϫ1 measured for PPase at 25°C in this work is slightly larger than a literature value of 290 s Ϫ1 (18). PP i Hydrolysis in DMSO-Because transfer of the substrate from bulk water to nonpolar solvents greatly accelerates the second-order rate constant for hydrolysis of phosphate monoesters and diesters (27,28), we sought to determine whether desolvation might also increase the rate of PP i hydrolysis. PPase has an unusually polar active site (10), so we chose to model PP i desolvation in the active site of PPase by transferring PP i from bulk water to a polar aprotic solvent, DMSO.…”

Preferred dissociative mechanism of phosphate monoester hydrolysis in low dielectric environments

“…The above preference for the stepwise mechanism may be relevant for the experimental observation [5,6] that the rate of hydrolysis is significantly enhanced in low dielectric media. For example, Stockbridge and Wolfenden [5] demonstrated that the hydrolysis of neopentyl phosphate dianion ðNP 2À Þ proceeds orders of magnitude faster in wet cyclohexane than in aqueous solution. This rate enhancement corresponds to a decrease in activation free energy by 11-12 kcal/mol [5].…”

“…For example, Stockbridge and Wolfenden [5] demonstrated that the hydrolysis of neopentyl phosphate dianion ðNP 2À Þ proceeds orders of magnitude faster in wet cyclohexane than in aqueous solution. This rate enhancement corresponds to a decrease in activation free energy by 11-12 kcal/mol [5]. The above rate enhancement is consistent qualitatively with the dramatic decrease in free energy barrier shown in Figure 3.…”

“…Regarding the comparison with the experiment by Stockbridge and Wolfenden [5], there are several points that require further consideration. For example, they demonstrated that the first-order hydrolysis rate of NP 2À in wet cyclohexane increases linearly with increasing concentration of water in cyclohexane, which implies that the hydrolysis of NP 2À is bimolecular, and hence a dissociative mechanism of more concerted character might be operative.…”

“…We also focus our attention on the solvent effects, namely, how the dielectric constant of the solvent may affect the preference for one mechanism over another. Our interest in the solvent effects above comes from a recent experiment by Stockbridge and Wolfenden [5], who demonstrated that the hydrolysis of neopentyl phosphate dianion in wet cyclohexane proceeds more than 10 8 times faster than in aqueous solution. Similar enhancement of phosphate hydrolysis rate is known for nitrophenyl phosphate dianion in DMSO/water mixture [6].…”