Purification and properties of the phosphofructokinase from Lactobacillus bulgaricus

Abstract

Phosphofructokinase, the enzyme which catalyzes the conversion of fructose 6-phosphate into fructose 1,6-bisphosphate in Lactobacillus buigaricus (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 stearotherrnophiius, 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 phosphoenoipyruvate. Partial inhibition by phosphoenoipyruvate 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 V,,,. Protection against thermal denaturation shows that the enzyme binds phosphoenofpyruvate 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.L . bulgaricus is one of the bacteria used by the food industry for large-scale fermentation of milk into cheese and yoghurt, but despite its economic importance, little is known of its biochemistry. L. bulgaricus has a simplified catabolism: (a) it can utilize only three sugars (glucose, fructose and lactose) as a carbon-energy source, (b) it is a strict fermenting bacterium which lacks a respiratory chain, (c) its fermentation produces almost exclusively lactic acid [ 11. L . buigaricus appears then to produce most (if not all) of the energy required for its growth solely by the glycolytic breakdown of sugars.In many organisms, the flux through the glycolytic pathway is, at least in part, controlled at the level of the reaction:ATP +fructose 6-phosphate --f ADP + fructose 1,B-bisphosphate, catalyzed by phosphofructokinase. Phosphofructokinases from a variety of sources are indeed endowed with rather sophisticated regulatory properties, with a marked cooperative behavior and/or potent allosteric effectors [2]. Also, one form of this enzyme appears to be rather conserved

Identification of a novel operon in Lactococcus lactis encoding three enzymes for lactic acid synthesis: phosphofructokinase, pyruvate kinase, and lactate dehydrogenase

“…The measured sizes (in kilobase pairs; expected sizes on the basis of the data in Fig. 2 are given in parentheses) of the hybridizing fragments were as follows: EcoRI, 2.05 (2.05 and 6.0); HindIII bulgaricus (Ldb) (25), Thermus thermophilus (Tth) (49), E. coli phosphofructokinase 1 (Eco) (22), B. stearothermophilus (Bst) (18), and L. lactis (LU) (this study). The first amino acids shown for the H. sapiens and S. cerevisiae enzymes are residues 14 and 204 in their respective published sequences, and only the first 38 residues have been reported for L. delbrueckii.…”

The genes for phosphofructokinase and pyruvate kinase of Lactobacillus delbrueckii subsp. bulgaricus constitute an operon

“…This need for fine differential control of Pfk and Pyk could explain why these enzymes are allosteric in most organisms (6). In L. bulgaricus, the regulation of Pfk and Pyk is coordinated, since the substrate of one enzyme is an allosteric effector of the other (15,17). A change in the concentration of one metabolite, either fructose-6-phosphate or PEP, will thus affect the activities of both enzymes.…”

The cooperativity and allosteric inhibition of Escherichia coli phosphofructokinase depend on the interaction between threonine-125 and ATP.

“…Indeed, the PFKs from Lactobacillus bulgaricus and Spiroplasma citri, which do not undergo an R --T transition (27,29) have, respectively, Asp and Ile at position 241 (22,23) while Glu-241 is present in the PFKs from E. coli (20), B. stearothermophilus (19), and Thermus thermophilus (22), which respond to PEP binding by an R --T transition (3,12,30). It is more difficult to predict the effect of a mutation of Arg-72 since this residue is engaged in an ionic bond in both the R and T states, albeit with different partners.…”