The rate of enzymic hydrolysis of phosphoric esters by prostatic phosphatase.

Delory & King (1943) published a comparative study of the hydrolysis by faecal alkaline phosphatase of a series of phosphoric esters and established a correlation between the pK values of their second phosphate dissociations, the optimum pH for the enzymic hydrolysis and the Michaelis constants of the esters. They showed that the more acid this phosphate dissociation, the more alkaline was the pH optimum, the greater was the amount of hydrolysis and the lower the Michaelis constant. They found their results consistent with the Martland & Robison (1927) theory of enzyme-substrate combination and with Kay's (1932) interpretation of the pH optima of alkaline phosphatase for different substrates as a balance between increase in the rate of hydrolysis and increase in the rate of enzyme inactivation with increasing alkalinity. Two more esters, the oand p-carboxyphenyl phosphates have been added to this series. For their synthesis the carboxyl groups of salicylic and phydroxybenzoic acid had to be protected by esterification before phosphorylation of the phenolic hydroxyl group by phosphorus oxychloride. Even then the usual conditions employed for this reaction (King & Nicholson, 1939) were too drastic, and the reaction mixture had to be kept at less than 100 throughout the phosphorylation. The crystalline esters so obtained had a much lower barium and higher phosphorus content than was expected; and when potentiometric titration showed only one titrable hydrogen per atom of phosphorus, and that with apK ofabout 1, it was realized that the product was a pyrophosphoric ester and not an orthophosphate. Only later was it discovered that the conditions we had been forced to adopt for the phosphorylation were almost exactly those described by Neuberg & Wagner (1926) for the synthesis of diphenyl pyrophosphate. Mild alkaline hydrolysis of these pyrophosphates split the pyrophosphate link, and set free the carboxyl group. The carboxyphenyl phosphates were isolated as barium salts; and titration now showed the three dissociations expected, which were ascribed in order ofdecreasing acidity to the primary phosphate, the carboxyl group and the secondary phosphate dissociations (Table 1).