2,3-Diphosphoglycerate Phosphatase from Human Erythrocytes

Abstract

Abstract 2,3-Diphosphoglycerate phosphatase has been purified from human red blood cells. It has been separated from phosphoglycerate mutase and acid phosphatase. The reaction catalyzed is 2,3-PGA + H2O → 3-PGA + Pi. In the absence of activators, 1 mµmole of substrate can be converted to products per min at 37° by enzyme from 1 ml of packed cells. The activity is increased by certain anions. There is activation by chloride or phosphate individually, which is enhanced by the simultaneous presence of both anions. In the presence of 0.1 m chloride, phosphate activates with a Ka of 0.7 mm. Fluoride or bromide, but not iodide, and bicarbonate can substitute for chloride. Anions that can substitute for phosphate and activate in the presence of chloride include arsenate, fluorophosphate, and sulfite. The most potent activator found is glycolate-2-P which can give a 1600-fold stimulation. In the presence of this activator, chloride is an inhibitor with respect to glycolate-2-P. Phosphate is a competitive inhibitor with respect to glycolate-2-P and the inhibition is related to the square of the phosphate concentration. The monophosphoglycerates are competitive inhibitors with respect to phosphate with enzyme activated by phosphate and chloride with a Ki of 3 µm. Monophosphoglycerates are competitive with respect to glycolate-2-P with enzyme activated by glycolate-2-P and chloride with a Ki of 2 µm. Glycine or taurine (at high pH) or pyrophosphate are activators in the presence of which there is inhibition by chloride. The Km of 2,3-diphosphoglycerate depends on the conditions of activation, varying from 0.08 µm with phosphate and 0.1 m chloride activation to 25 µm with glycolate-2-P and 0.1 m chloride. The apparent Vmax varies over a 500-fold range. Phosphoglycerate increases the Km of 2,3-PGA and inhibits with a Ki of 6 µm. The maximal velocity of the enzyme is obtained at pH 6.4 with either phosphate and chloride or glycolate-2-P as activators. With fluorophosphate and chloride, the optimum rate is at pH 6. It is suggested that the phosphate and phosphoglycerate levels are important for determining the rate of hydrolysis of 2,3-diphosphoglycerate in the human red blood cell.