Kinetics and subunit interaction of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system.
Purified mannitol-specific enzyme II (EIImtl), in the presence of the detergent Lubrol, catalyzes the phosphorylation of mannitol from P-HPr via a classical ping-pong mechanism involving the participation of a phosphorylated EIImtl intermediate. This intermediate has been demonstrated by using radioactive phosphoenolpyruvate. Upon addition of mannitol, at least 80% of the enzyme-bound phosphoryl groups can be converted to mannitol 1-phosphate. The EIImtl concentration dependence of the exchange reaction indicates that self-association is a prerequisite for catalytic activity. The self-association can be achieved by increasing the EIImtl concentration or at low concentrations of EIImtl by adding HPr or bovine serum albumin. The equilibrium is shifted toward the dissociated form by mannitol 1-phosphate, resulting in a mannitol 1-phosphate induced inhibition. Mannitol does not affect the association state of the enzyme. Both mannitol and mannitol 1-phosphate also act as classical substrate inhibitors. The apparent Ki of each compound, however, is approximately equal to its apparent Km, suggesting that mannitol and mannitol 1-phosphate bind at the same site on EIImtl. Due to strong inhibition provided by mannitol and mannitol 1-phosphate in the exchange reaction, the kinetics of this reaction cannot be used to determine whether the reaction proceeds via a ping-pong or an ordered reaction mechanism.
D002250:Carbon Radioisotopes; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008353:Mannitol / Q000378:metabolism; D008355:Mannitol Phosphates / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid
D002250:Carbon Radioisotopes; D046911:Macromolecular Substances; D008355:Mannitol Phosphates; D010761:Phosphorus Radioisotopes; D011773:Pyruvates; C030070:mannitol-1-phosphate; D008353:Mannitol; D010728:Phosphoenolpyruvate; D019289:Pyruvic Acid; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C037992:phosphoenolpyruvate-mannose phosphotransferase