PH-dependent oxygen exchange of phosphate and hydrolysis (of sulfur) from thiophosphate catalyzed by acid phosphatases

Abstract

Phosphate oxygen exchange and thiophosphate hydrolysis reactions catalyzed by a number of homogeneous acid phosphatase isoenzymes were studied as a function of pH. Data are presented for the pH dependence of the phosphate oxygen—water oxygen exchange reaction catalyzed by human prostatic acid phosphatase. The K cat for phosphate oxygen exchange is approximately constant from pH 2.7 to 7.0 and declines at high pH. This pH dependence resembles that of V for typical substrate hydrolysis reactions. Human prostatic acid phosphatase is shown to catalyze the hydrolysis of inorganic thiophosphate to phosphate ion. The rate of thiophosphate ion hydrolysis is maximal in the pH range 3 to 5 and decreases at higher pH. Hydrolysis reactions of phosphomonoesters and related phosphorothioatemonoesters were compared using acid phosphatases from animal and plant sources. Wheat germ acid phosphatase catalyzes the hydrolysis of adenosine 5′-phosphorothioate and p-nitrophenyl phosphorothioate, though at a much slower rate than the hydrolysis of the corresponding phosphomonoesters. Similarly, human prostatic acid phosphatase hydrolyses adenosine 5′-phosphorothioate at a very slow rate ( k cat = 2.3 × 10 −2s −1), whereas the corresponding phosphomonoester is hydrolyzed four orders of magnitude more rapidly. A human liver acid phosphatase isoenzyme hydrolyses AMP and p-nitrophenyl phosphate at a rate of 1.0 and 1.2 × 10 2 s −1, respectively, but adenosine 5′-phosphorothioate and p-nitrophenyl phosphorothioate are not detectably hydrolyzed by this enzyme. A low-molecular-weight acid phosphatase from bovine liver hydrolyzed p-nitrophenyl phosphate at a rate of 35 s −1, but it failed to hydrolyze AMP, adenosine 5′-phosphorothioate, or p-nitrophenyl phosphorothioate. Various thermodynamic parameters for phosphate oxygen exchange and for the hydrolysis of thiophosphate and p-nitrophenyl phosphate catalyzed by human prostatic acid phosphatase were also measured. The activation energy for phosphate oxygen exchange catalyzed by human prostatic acid phosphatase is 17 kcal mol −1 while the activation energy for the hydrolysis of p-nitrophenyl phosphate is 10.9 kcal mol −1. K m(app) for the hydrolysis of p-nitrophenyl phosphate by human prostatic acid phosphatase decreases from 0.61 m m at 7 °C to 0.10 m m at 37 °C.