MECHANISTIC SIGNIFICANCE OF PHOSPHATE LABELING OF ALKALINE PHOSPHATASE

Alkaline phosphatase reacts with orthophosphate at pH <8 to form a thermodynamically stable phosphoprotein. In order to study this phosphoprotein, a rapid quenching technique in conjunction with scintillation counting was developed which allowed for an accurate determination of amount of phosphoprotein formed. The reaction was stopped in 6 M perchloric acid by forcing 0.5 ml/sec of the solution through a 0.020-i.d. capillary tip into the quenching solution by means of a motor-driven syringe. The covalent labeling was measured from pH 8.0 to 5.0 and at different concentrations of phosphate. In this way the equilibrium constants for the dissociation of the enzyme-phosphate addition complex at 26° (Michaelis complex) were found to be 4.1 X 10-6 at pH 7.0, 6.7 X 10-6 at pH 6.0, and 5.4 X 10-5 at pH 5.5, while the equilibrium constants for the hydrolysis of the covalent phosphoprotein were found to be 1.4 X 10-4 at pH 7.0, 6.0 X 10-6 at pH 6.0, and 1.2 X 10-5 at pH 5.5. Measurements were also made at 3 °. These constants are in good agreement with the values of these quantities for the phosphoryl-enzyme intermediate determined previously by kinetic methods. Similarly the calculated kinetic inhibition constants for phosphate at 26°, 3.8 X 10-6 at pH 7.0, 3.1 X 10-6 at pH 6.0, and 1.0 X 10-5 at pH 5.5, agreed with the experimental values. Thus the properties of the kinetically deduced phosphoryl-enzyme intermediate are the same as the properties of the phosphoprotein obtained in labeling experiments so that there is good reason to conclude that the two are the same. The phosphoprotein is stable relative to free enzyme and inorganic phosphate at all pH's studied, yet very little is formed at pH 7.0 and 8.0. The reason for this seeming anomaly is that the addition complex between enzyme and phosphate is even more stable at these pH's. As the pH is lowered, the relationship changes so that at pH 5.5 the covalent phosphoryl-enzyme is much more stable than the enzyme-phosphate complex and labeling is extensive. © 1969, American Chemical Society. All rights reserved.