Preparation and properties of a phosphoenzyme from the phosphoglucomutase of Micrococcus lysodeikticus.
1. Phosphoglucomutase from Micrococcus lysodeikticus was incubated with (14)C- and (32)P-labelled glucose 1,6-diphosphate and separated from the cofactor on a Sephadex column. (32)P-labelled phosphate (0.7mol/mol of enzyme) was associated with the enzyme, but no (14)C label was. 2. The (32)P-labelled enzyme exchanged its label with the substrates. When the labelled enzyme was incubated in Tris buffer, pH8.3, at 30 degrees C the proportion of exchangeable label slowly fell indicating a half-life of the phosphoenzyme of about 50h. 3. When HClO(4) was added to the labelled phosphoenzyme all of the label was precipitated with the protein and none was released as P(i). On alkaline hydrolysis P(i) was released at a rate comparable with the rate of hydrolysis of the phosphoenzyme from rabbit muscle. 4. We conclude that the phosphoenzyme from Micrococcus lysodeikticus yields a relatively stable, catalytically active phosphoenzyme when treated with cofactor, and that there is no evidence for the formation of an enzyme-glucose 1,6-diphosphate complex. The properties of the phosphoenzyme, which resemble those of rabbit muscle phosphoglucomutase, suggest that the phosphate may be bound to serine.
D000818:Animals; D001665:Binding Sites; D002250:Carbon Radioisotopes; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005958:Glucosephosphates / Q000378:metabolism; D006207:Half-Life; D007700:Kinetics; D008837:Micrococcus / Q000201:enzymology*; D008970:Molecular Weight; D010710:Phosphates; D010733:Phosphoglucomutase / Q000302:isolation & purification* / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification*; D010761:Phosphorus Radioisotopes; D011817:Rabbits; D012694:Serine / Q000378:metabolism; D013997:Time Factors
D002250:Carbon Radioisotopes; D005958:Glucosephosphates; D010710:Phosphates; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D012694:Serine; D010733:Phosphoglucomutase