Purification and characterization of glpQ-encoded glycerophosphodiester phosphodiesterase from Escherichia coli K-12.
Periplasmic glycerophosphodiester phosphodiesterase (EC 3.1.4.2) of Escherichia coli was purified seven-fold to near homogeneity from the cold osmotic shock fraction of a strain harboring a multicopy plasmid carrying the glpQ gene. The enzyme had a minimum subunit molecular weight of 40,000 as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native size of the enzyme was 70,000 as assessed by gel filtration chromatography and 75,000 as assessed by nondenaturing gradient polyacrylamide gel electrophoresis, indicating that the native state of the enzyme is dimeric. The enzyme hydrolyzed the deacylation products of all glycerophospholipids tested including glycerophosphocholine, glycerophosphoethanolamine, glycerophosphoglycerol, glycerophosphoinositol, and glycerophosphoserine. The enzyme did not release glycerol or sn-glycerol 3-phosphate from phosphatidyl-DL-glycerol or lysophosphatidyl-DL-glycerol present in Triton X-100 micelles. The enzyme functioned optimally at pH 7.8. The enzyme was totally inactivated by dilution into 1 mM ethylenediaminetetraacetate or ethylene glycol bis(beta-aminoethyl ether)-N,N-tetraacetic acid. Activity was restored by the addition of Ca2+ or Cd2+, and was partially restored by the addition of Mn2+ or Cu2+. Co2+, Mg2+, Zn2+, and Fe2+ did not restore activity. The presence of calcium ions decreased the Km of the enzyme for the substrate, glycerophosphoglycerol, and increased the Vmax.
Larson
T J
TJ
Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg 24061.
van Loo-Bhattacharya
A T
AT
D002104:Cadmium / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent; D002850:Chromatography, Gel; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D002413:Cations, Divalent; D046911:Macromolecular Substances; D002104:Cadmium; D004533:Egtazic Acid; D004492:Edetic Acid; D010727:Phosphoric Diester Hydrolases; C038133:glycerophosphodiester phosphodiesterase; D002118:Calcium