Phosphoryl Transfer from S‐Substituted Monoesters of Phosphorothioic Acid to Various Acceptors Catalyzed by Alkaline Phosphatase from Escherichia coli

Alkaline phosphatase ( Escherichia coli ) was found to catalyze the synthesis of monophosphate esters of serine, ethanolamine, propanolamine, butanol, glycerol, l ‐glucose, and tris(hydroxymethyl)aminomethane by phosphoryl transfer from cysteamine S ‐phosphate (donor) to the respective alcohols (acceptors). Maximum enzymic synthesis of the above esters occurred at pH 7.8. The newly‐formed phosphate esters were measured quantitatively after separation of the products by the aid of paper high voltage electrophoresis techniques or by an amino acid analyzer. The percentage of the enzyme‐catalyzed synthesis of the new phosphate esters varied from 15–39% using different acceptors under otherwise identical experimental conditions. It is pertinent to note that the same compounds were phosphorylated to essentially the same extent when the donor (substrate) compound was serine O ‐phosphate, aminoethanol O ‐phosphate or p ‐nitrophenyl O ‐phosphate. The rates of enzymic consumption of cysteamine S ‐phosphate ( v h + v a ) were measured at different pH values and at different concentrations of acceptor (Tris or aminoethanol) in the presence of 1.5 M NaCl using 3 mM substrate (cysteamine S ‐phosphate). The presence of either Tris or aminoethanol gave the same pH profile for the rate of consumption ( v h + v a ) of cysteamine S ‐phosphate with a maximum value around pH 7.8. This value differs from that found for the same reaction in barbital buffer (pH 9.0). The rate of enzymic consumption of cysteamine S ‐phosphate was also measured at various substrate and Tris concentrations at pH 7.8 under constant ionic strength ( T/2 = 1.0). The same K m value (0.24 mM) was obtained at Tris concentrations varying from 0.02 M to 0.5 M. The V max values derived from these experiments were linearly related to the Tris concentrations up to 0.5 M Tris. The pH profile of the rate of consumption of p ‐nitrophenyl phosphate as well as the K m value (0.26 mM) were similar to the corresponding values obtained for cysteamine S ‐phosphate. The rate of hydrolysis ( v h ) could not be measured when cysteamine S ‐phosphate served as the donor compound due to the instability of cysteamine S ‐phosphate under the conditions required for inorganic phosphate assay. Therefore, the rate of transfer ( v a = v RSH – v p 1 ) could not be estimated. The pH dependence of the rate of transfer, v a , was calculated from experimental data obtained when p ‐nitrophenyl phosphate served as the donor compound and Tris served as the acceptor. The pH profile for the rate of transfer, v a , obtained from these measurements was very similar to that obtained by direct measurement of Tris O ‐phosphate formation using cysteamine as the donor. It is suggested that phosphoryl transfer occurs through a phosphorylated enzyme intermediate, and therefore, the types of compounds that could serve as acceptors are independent of the donor compounds and depend only on the bond energy of the particular phosphorylated enzyme intermediate.