Phosphoenolpyruvate Synthetase of Escherichia coli: PURIFICATION, SOME PROPERTIES, AND THE ROLE OF DIVALENT METAL IONS

Abstract

A procedure for the preparation of highly purified phosphoenolpyruvate synthetase from Escherichia coli is described. The purified enzyme is markedly stabilized against inactivation by high concentrations of sucrose and is susceptible to inactivation by sulfhydryl reagents. The binding of the activating manganous ion to the dephosphoenzyme was investigated by electron spin resonance and proton relaxation rate of water. The proton relaxation rate of water was enhanced 12 times relative to the Mn2+ aquo-ion. Addition of pyruvate decreased the enhancement, indicating the formation of a dephosphoenzyme-manganese-pyruvate complex. On the basis of a molecular weight of 250,000 it was calculated that 3 to 4 moles of Mn2+ were bound per mole of enzyme with K d ≅ 30 µ m . The phosphate content of phosphoenzyme whether prepared from ATP or phosphoenolpyruvate was 1 to 2 moles of phosphate per mole of enzyme. Initial velocity studies of the forward reaction indicate that an irreversible step occurs between the binding of MgATP to the enzyme and the binding of pyruvate. MgATP and pyruvate combine with the dephospho- and the phosphoenzyme form, respectively, to form kinetically active complexes; the Michaelis constants are K m g atp = 28 µ m and Kpyruvate = 20 µ m at 5 m m free Mg2+. A requirement for free divalent metal ion (Mg2+ or Mn2+) in addition to that bound to ATP was indicated by the kinetic studies and was implicated in the binding of pyruvate to the enzyme. At high concentrations of free Mg2+ and Mn2+, the reaction is inhibited. Ca2+ is not an activator but inhibits the Mn2+-activated reaction.