title: Purification and characterization of the Tetrahymena pyriformis P-C bond forming enzyme phosphoenolpyruvate phosphomutase.
abstract: In this paper the purification and characterization of the Tetrahymena pyriformis enzyme phosphoenolpyruvate phosphomutase are described. PEP phosphomutase was first fractionated from T. pyriformis cellular extract by using 70% ammonium sulfate. Chromatography of the crude protein fraction on a DEAE-cellulose column followed by phenyl-Sepharose column chromatography and then Bio-Gel P-200 column chromatography afforded pure PEP phosphomutase in an approximate overall yield of 70 units/150 g of cells. The maximum turnover number observed for PEP phosphomutase catalysis of the phosphonopyruvate----PEP reaction is 38 s-1 (25 degrees C). The enzyme was shown to be a homodimer of 38,000-dalton subunits and to require a divalent metal ion for activity. Mg2+ (relative Vm = 1), Co2+ (rel Vm = 0.5), Zn2+ (rel Vm = 0.4), and Mn2+ (rel Vm = 0.3) each satisfied the cofactor requirement. Binding of the physiological cofactor, Mg2+ (Ki = 0.3 mM at pH 7.5), and phosphonopyruvate (Km = 2 microM at pH 7.5) was found to be ordered, with cofactor binding preceding substrate binding. Within the pH range of 5-9 catalysis (Vm) was found to be pH independent, while phosphonopyruvate binding dropped at acidic and basic pH.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
authors.forename: E D
authors.initials: ED
authors.lastname: Bowman
authors.forename: M S
authors.initials: MS
authors.lastname: McQueney
authors.forename: J D
authors.initials: JD
authors.lastname: Scholten
authors.forename: D
authors.initials: D
authors.lastname: Dunaway-Mariano
mesh_terms: D000818:Animals; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008970:Molecular Weight; D017875:Phosphotransferases (Phosphomutases); D012995:Solubility; D013769:Tetrahymena pyriformis / Q000201:enzymology*
chemical_list: D008670:Metals; D007535:Isomerases; D017875:Phosphotransferases (Phosphomutases); C057167:phosphoenolpyruvate mutase