Characterization of a phosphoenzyme intermediate in the reaction of phosphoglycolate phosphatase.
When 32P-glycolate and phosphoglycolate phosphatase from spinach are mixed, 32P is incorporated into acid precipitated protein. Properties that relate the phosphorylation of the enzyme to the phosphatase are: the Km value for P-glycolate is similar for protein phosphorylation and substrate hydrolysis; the 32P in the phosphoenzyme is diluted by unlabeled P-glycolate or the specific alternative substrate, ethyl-P; the activator Cl- enhances the effectiveness of ethyl-P as a substrate and as an inhibitor of the formation of 32P-enzyme; and 32P is lost from the enzyme when 32P-glycolate is consumed. The phosphorylated protein has a molecular weight of 34,000, which is half that of the native protein and is similar in size to the labeled band that is seen on sodium dodecyl sulfate-polyacrylamide gels. The enzyme-bound phosphoryl group appears to be an acylphosphate from its pH stability, being quite stable at pH 1, less stable at pH 5, and very unstable above pH 5. The bond is readily hydrolyzed in acid molybdate and it is sensitive to cleavage by hydroxylamine at pH 6.8. The demonstration of enzyme phosphorylation by 32P-glycolate resolves the dilemma presented by initial rate studies in which alternative substrates appeared to have different mechanisms (Rose, Z. B., Grove, D. S., and Seal, S. N. (1986) J. Biol. Chem. 261, 10996-11002).
Seal
S N
SN
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Rose
Z B
ZB
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D006016:Glycolates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010750:Phosphoproteins / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010944:Plants / Q000201:enzymology*; D011485:Protein Binding; D014316:Tritium
D006016:Glycolates; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D014316:Tritium; C022408:phosphoglycolate phosphatase; D010744:Phosphoric Monoester Hydrolases; C013193:phosphoglycolate