Formation of an active phosphoenzyme by diphosphoglycerate-dependent phosphoglyceromutases from muscle, kidney and yeast

Abstract

1. 1. The reactions of d2,3-diphosphoglycerate with phosphoglyceromutases (2,3-diphosho- d-glycerate:2-phospho- d-glycerate phosphotransferase. EC 2.7.5.3) from rabbit muscle, yeast and pig kidney have been examined by observing spectrophotometrically the formation of monophosphoglycerate in the presence of excess enolase (EC 4.2.1.11), pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27). Monophosphoglycerate was produced in an initial burst and thereafter in a steady rate until hydrolysis of 2,3-diphosphoglycerate was complete. The initial burst was interpreted as the formation of a phosphoenzyme (2 moles phosphate per mole enzyme in rabbit muscle and pig kidney; >3 moles phosphate per mole yeast enzyme) and successive additions of 2,3-diphosphoglycerate indicated that the phosphoenzymes were unstable with half-lives of the order of 5, 8 and 1–2 min for rabbit muscle, pig kidney and yeast, respectively. The 2,3-diphosphoglycerate phosphatase (EC 3.1.3.1.3) activity could be attributed to the instability of the phosphoenzymes. The K m values for 2,3-diphosphoglycerate were low ( ⪕ 1 μ M ). 2. 2. The reactions of 2,3-diphosphoglycerate with the enzymes from rabbit muscle and pig kidney were also examined spectrophotometrically in the presence of rate-limiting quantities of enolase. The initial velocities indicated that little, if any, monophosphoglycerate associated with the phosphoenzymes to form enzyme-2,3-diphosphoglycerate complexes at monophosphoglycerate concentrations of about 11μM. With rapid dialysis the dephosphoenzymes were shown not to bind monophosphoglycerate under these conditions. 3. 3. The rabbit muscle, pig kidney and yeast enzymes are shown to obey Michaelis-Menten kinetics down to 3-phosphoglycerate concentrations of the order of 1 μM. In view of the evidence that little, if any, of the enzyme-2,3-diphosphoglycerate species is present in solution under these conditions, and since there is no activation in the enolase coupled mutase assay as d-2-phosphoglycerate accumulates, it is concluded that the phosphoenzymes must themselves be catalytically active. The possibility that enzyme-2,3-diphocphoglycerate complexes if they exist, may also be catalytically active is not excluded. 4. 4. After incubation with 14C- and 32P-labelled d-2,3-diphosphoglycerate and gel filtration the enzymes were obtained labelled with 32P but not with 14C. 2 moles phosphate per mole enzyme were bound by rabbit and pig kidney. The 32P bound to the enzymes exchanged with the substrate and was liberated as P i with HClO 4. After denaturation with sodium dodecyl sulphate, the phosphoproteins from yeast and pig kidney were stable to alkali by hydrolysed by acid suggesting the involvement of phosphohistidine. 5. 5. The evidence presented for the formation of active phosphoenzymes supports the hypothesis that d-2,3-diphosphoglycerate-dependent phosphoglyceromutases are closely related to d2,3-diphosphoglycerate diphosphatases.