PMID: 2121271Jul 31, 1990Paper

Purification and characterization of the Tetrahymena pyriformis P-C bond forming enzyme phosphoenolpyruvate phosphomutase

Biochemistry
E D BowmanD Dunaway-Mariano

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.

References

Oct 10, 1977·Biochemical and Biophysical Research Communications·M H Klapper
May 9, 1967·Biochimica Et Biophysica Acta·A Trebst, E Pistorius
Jun 5, 1984·Biochemistry·V E AndersonW W Cleland
Sep 19, 1959·Nature·M HORIGUCHI, M KANDATSU

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