A Phosphoenzyme Intermediary in Phosphoglycerate Kinase Action

Abstract

Rabbit muscle phosphoglycerate kinase (EC 2.7.2.3) has been phosphorylated with γ-32P-ATP and then isolated by gel filtration. Phosphorylation was favored by the presence of phosphoenolpyruvate and a catalytic amount of pyruvate kinase, which appeared to remove ADP from the phosphorylation equilibrium. The phosphoenzyme was catalytically active, transferring its phosphoryl group to ADP to synthesize ATP. The phosphoenzyme also transferred its phosphoryl group to 3-phosphoglycerate to form 1,3-diphosphoglycerate, which, being unstable, was not isolated. It was converted instead to the corresponding hydroxamate or transformed enzymatically to fructose 1,6-diphosphate. Inorganic phosphate was released from the phosphoenzyme by neutral hydroxylamine at room temperature within 10 min. The pH stability profile of the phosphoenzyme is U-shaped. These facts accord with a covalent binding of the phosphoryl group to a carboxyl of the enzyme.