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Purification and properties of the phosphofructokinase from Lactobacillus bulgaricus. A non-allosteric analog of the enzyme from Escherichia coli.

Phosphofructokinase, the enzyme which catalyzes the conversion of fructose 6-phosphate into fructose 1,6-bisphosphate in Lactobacillus bulgaricus (Lactobacillus delbrueckii, subspecies bulgaricus) has been purified to homogeneity and some of its structural and functional properties have been studied. The enzyme is a tetramer composed of four 35-kDa subunits. Its N-terminal sequence determined on 38 residues is homologous to those of the major allosteric enzymes from Escherichia coli and Bacillus stearothermophilus, suggesting that the three proteins have closely related structures. The maximum velocity of the enzyme from L. bulgaricus increases with pH according to the ionization of a group with a pK of 6.2. At all pH values, the saturation by fructose 6-phosphate is hyperbolic. At the optimum pH of 8.2, the maximum velocity and the affinities for the ATP and fructose 6-phosphate substrates are not modified by the presence of ADP or GDP nor by phosphoenolpyruvate. Partial inhibition by phosphoenolpyruvate exists at acidic pH, but is not related to an allosteric mechanism similar to that in E. coli. This inhibition results from a shift from 6.2 to 7.1 of the pK of an ionizable group which controls Vmax. Protection against thermal denaturation shows that the enzyme binds phosphoenolpyruvate and not GDP. The phosphofructokinase from L. bulgaricus appears as a structural analog of the E. coli enzyme which does not undergo an allosteric transition between two states R and T, but instead remains in a unique conformational state, intermediate between the R and T states; the active sites have an R-like conformation since they bind fructose 6-phosphate, whereas the regulatory sites have a T-like conformation since they bind phosphoenolpyruvate and not GDP.