Abstract
The observed equilibrium constants for hydrolysis (Kobs) of a phosphoester and a phosphoanhydride bond were measured under a variety of conditions likely to alter the interactions of reactants and products with water. These included increasing the pH of the medium from 5.0 to 10.0, increasing the MgCl2 concentration form 0 to 200 mM, and decreasing the water activity of the medium by adding either dimethyl sulfoxide (50%, v/v) or polyethylene glycol 6,000-8,000 (50%, w/v). The Kobs for phosphoesters such as phosphoserine, glucose phosphate, glycerol phosphate, and ethylene glycol phosphate varied little over this wide range of conditions, the extreme values of Kobs being 12 and 200 M. In contrast, the Kobs for the phosphoanhydride bond of pyrophosphate varied from a value greater than 20,000 to 0.1 M. In totally aqueous media at a pH between 7.0 and 8.0 and in the presence of 0.5-1.0 mM MgCl2, the energy of hydrolysis of pyrophosphate was 1.2-4.0 kcal/mol greater than that of phosphoserine. However, when the water activity was decreased by adding polyethylene glycol to the medium within the same pH and MgCl2 concentration range, the energy of hydrolysis of phosphoserine became 2.0-2.5 kcal/mol greater than that of pyrophosphate. The results suggest that for phosphoesters, the solvation energies of reactants and products, unlike the case of phosphoanhydride bonds, are not the major factors in determining the energy of hydrolysis.
Highlights
The observed equilibrium constants for hydrolysis support this proposal. ( a ) Theoretical calculations indicate (Kob)of a phosphoester and a phosphoanhydride bond that for some phosphorylated compounds, the free energy of were measured under a varietyof conditions likely to hydrolysis in gaseous phase differs greatly from that deteralter the interactions of reactants and products with water
Since solvation energy of both pyrophosphate andPi varies greatly depending on the ionic species formed (1,2), these effects of K0bcan be ascribed to changes in solvation energy of both reactants and products (1,2, 5 ) . (c) The Kob of pyrophosphate measured in totally aqueous media is higher than that determined in the presence of organic solvents or agentsthat alter thwe ater structure, such as polymers of ethylene glycol (5,8)
The results and in thepresence of 0.5-1.0 mM MgC12, the energy indicate that solvation energy does not play a major role in of hydrolysis of pyrophosphate was 1.2-4.0 kcallmol determining the free energy of hydrolysis of phosphoester greater than that of phosphoserine
Summary
Criteria for Equilibrium-Alkaline phosphatase catalyzes the formation of phosphoester bonds from Pi and alcohols (9). The same amount of either pyrophosphate or phosphosernonradioactive phosphoserine (20 mM) to the aqueous phase ine was synthesized after 2 and 4 h using two different enzyme followed byhydrolysis of the mixture with alkaline phospha- concentrations. The various Kobe were calculated from the molar concentration of the radioactive material remaining in the aqueous phase and theamount of reactants as follows:for pyrophosphate, Kobe was attained using yeast inorganic pyrophosphatase and con- = (Pi)'/(PPi) and for phosphoserine, Kok = (Pi) (serine)/(phosphoditions similar to those used for phosphoserine. Synthesis of phosphoserine was measured in media containing 100 mM MOPS/KOH buffer (pH 7.0), 200 mM I,(+)-serine, 1mM [32P]Pi,[5] pg/ml alkaline phosphatase, and either no added MgCl, (A) or 100 mM MgClz (0).Essentially the same amount of radioactive pyrophosphate was synthesized after 2 and 4 h incubation at thedifferent temperatures shown in the Figure.
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