Phosphoryl transfer reactions solvent effects

Appleyard (64) noted that addition of ethanol to incubation mixtures of sodium phenolphthalein diphosphate with prostatic extract increased the rate of free phenolphthalein formation. Phosphate ion failed to show a comparable increase, and this discrepancy was attributed to transphosphorylation. Phosphoryl transfer may be effected by prostatic phosphatase to acceptors other than solvent (65-67). Nigam and Fishman (25) studied phosphoryl transfer under conditions of 60-80% transfer to an acceptor. In the case of 1,4-butanediol, the optimal concentration was 0.8 M. In this experiment, water molecules outnumbered acceptor molecules by 55/0.8 or 70-fold. In spite of this, transfer far exceeded hydrolysis. Phosphoryl transfer to aliphatic alcohols can be easily measured when phosphates are used as donor compounds. The difference between alcohol formation from the substrate and phosphate ion production is a measure of the transfer reaction. Table IX (25) shows that four different substrates can transfer phosphoryl to butanediol with high efficiency. Table X (25) shows that aliphatic alcohols are good acceptors... 

Metal Ion Effects. The metal ion effects on the acid-catalyzed hydrolysis of PPS also were examined by Benkovic and Hevey (5). However, they observed that in water near pH 3, the rate enhancement in the presence of an excess of metal ion was at most only threefold (Mg2+, Ca2+, Al3+) and in some cases (Zn2+, Co2+, Cu2+) the rate was actually retarded. We thought that the substrate PPS and Mg2+ ion should be hydrated heavily in water so that their complexa-tion for rate enhancement is weak. If, however, the hydrolysis is carried out in a solvent of low water content, such complexation would not occur, and therefore, the rate enhancement might be more pronounced. This possibility appears to be supported by the fact that the active sites of many enzymes are hydrophobic. Of course, there is a possibility that the S—O fission may not require metal ion activation. In this connection, it is interesting to note that in biological phosphoryl-transfer reactions the enzymes generally require divalent metal ions for activity (7, 8, 9), but such metal ion dependency appears to be less important for sulfate-transfer enzymes. For example, many phosphatases require metal ions, but no sulfatase is known to be metal... 

Solvent kinetic isotope effects (SKIEs) in H2O/D2O mixtures on the reaction of /)NPP catalyzed by calcineurin gave a small normal value of 1.35. Proton inventory and fractionation data are consistent with a mechanism involving a single proton transfer from a metal-bound water, although due to the small KSIE value and the inherent experimental error of the proton inventory technique, the participation of a second proton could not he excluded. Further information has been furnished by heavy-atom isotope effects. Reaction of NPP catalyzed by APP shows that phosphoryl transfer is fully rate limiting. However, for calcineurin the... 

To understand the progression of experiments that led to the discovery and interpretation of solvent effects in phosphoryl transfer, it is important to provide a brief overview of the mechanism of phosphoryl transfer in solution. In the past decade, two excellent reviews on the mechanism and catalysis of these reactions have been published, and I encourage the interested reader to obtain and review them. 

---