Purification and characterization of a small membrane-associated sugar phosphate phosphatase that is allosterically activated by HPr(Ser(P)) of the phosphotransferase system in Lactococcus lactis.
In the Gram-positive bacterium, Lactococcus lactis, nonmetabolizable cytoplasmic sugar phosphates, accumulated by the phosphoenolpyruvate:sugar phosphotransferase system, are rapidly dephosphorylated and expelled from the cell upon addition of glucose (inducer expulsion). Our recent studies have established that a metabolite-activated, ATP-dependent protein kinase that phosphorylates serine-46 in HPr of the phosphoenolpyruvate:sugar phosphotransferase system activates a sugar phosphate phosphatase, thus initiating the inducer expulsion process. A membrane-associated, HPr(Ser(P))-dependent phosphatase has been identified, solubilized from the membrane, separated from other cellular phosphatases, and purified to near homogeneity. It exhibits a low subunit molecular mass (10 kDa) and behaves on gel filtration columns like a monomeric enzyme. It has broad substrate specificity, optimal activity between pH 7.0 and 8.0, is dependent on a divalent cation for activity, and is not inhibited by fluoride. It is stimulated more than 10-fold by HPr(Ser(P)) or a mutant derivative of HPr, S46D HPr, in which the regulatory serine is changed to aspartate, which bears a permanently negative charge as does phosphate. Stimulation is due both to an increase in the maximal velocity (Vmax) and a decrease in the Michaelis-Menten kinetic constant (Km) for sugar phosphate. The enzyme exhibits a Ka for S46D HPr of 15 microM. Although the enzyme is thermally stable, activation by HPr(Ser(P)) is heat sensitive.
Ye
J J
JJ
Department of Biology, University of California at San Diego, La Jolla 92093-0116, USA.
Saier
M H
MH
D000494:Allosteric Regulation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D013294:Lactococcus lactis / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000502:physiology*; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D010744:Phosphoric Monoester Hydrolases; C022700:sugar-phosphatase; D008274:Magnesium