The Purification and Properties of Diphosphoglycerate Mutase from Human Erythrocytes

Abstract

Diphosphoglycerate mutase has been purified from the red blood cells of rabbits and humans. The enzyme is found only in the soluble fraction of human red cell hemolysates at an average level of 0.93 unit per ml of packed cells. The stoichiometry has been verified as: 1,3-PGA + 3-PGA → 2,3-PGA + 3-PGA where PGA is phosphoglyceric acid. The kinetic studies with the human enzyme indicate Km values of 0.53 µ M for 1,3-diphosphoglycerate and 1 to 1.5 µ M for 3-phosphoglycerate. 2,3-Diphosphoglycerate is a competitive inhibitor of 1,3-diphosphoglycerate with a Ki of 0.85 µ M ; it is noncompetitive with 3-phosphoglycerate. Inorganic phosphate inhibits competitively with 3-phosphoglycerate with a Ki of 314 µ M . The Km of 1,3-diphosphoglyceric acid is independent of the level of 3-phosphoglycerate. An ordered bimolecular reaction is proposed in which 1,3-diphosphoglycerate combines with free enzyme, followed by the combination with 3-phosphoglycerate to form a ternary complex. After conversion to the products, 3-phosphoglycerate is released before 2,3-diphosphoglycerate. Diphosphoglycerate mutase is inhibited by sulfhydryl reagents and purified fractions require the addition of mercaptoethanol for optimal activity. Phosphoglycolic acid is stimulatory at levels below 0.4 m M and inhibitory at higher levels. 2-Phosphoglycerate is an activator of the reaction. It decreases the Km of 3-phosphoglycerate and increases the over-all activity of the enzyme. 2-Phosphoglyceric acid also acts as a phosphate acceptor in the over-all reaction to produce 2,3-diphosphoglycerate.