Regulatory effects of potassium and inorganic anions on the NADP-specific malic enzyme of Escherichia coli.

Abstract

The effects of K+ and various anions on the catalytic and regulatory properties of the NADP-specific malic enzyme of Escherichia coli are reported. Studies on the susceptibility of the enzyme to proteolysis indicate that K+ binds directly to the enzyme with a resultant change in enzyme conformation. Kinetic studies indicate that the binding of optimal concentrations of K+ results in activation of the enzyme, increasing both the Vmax and the affinity of the enzyme for divalent cations. The inhibition of enzyme activity observed at KCl concentrations greater than 50 mM is shown to be nonspecific, resulting from increasing ionic strength. The mixed cooperativity between malate-binding sites previously reported at optimal K+ concentration is more pronounced at nonoptimal K+ concentrations (0 and 150 mM). The regulatory effect of metal cofactors and the mixed cooperativity between malate-binding sites is abolished when kinetic studies are conducted at low ionic strength or in the presence of acetate. Acetate appears to act as an activator, increasing the affinity of the enzyme for malate and protecting the enzyme against the inhibition caused by high ionic strength. It is postulated that the enzyme is operating in vivo in a partially inhibited state owing to the ionic strength of the cytoplasm. The kinetic studies conducted at higher ionic strength in vitro are therefore more applicable to the in vivo situation.