Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: equilibrium kinetics and mechanism of enzyme I phosphorylation

Abstract

The phosphorylation of enzyme I from the E. coli phosphoenolpyruvate-dependent phosphotransferase system was studied by isotope exchange between phosphoenolpyruvate and pyruvate. Experiments monitoring 1H-2H exchange showed that enzyme I phosphorylation is accompanied by the transfer of a proton from the enzyme to the C-3 atom of the substrate. 14C-12C-exchange experiments with both deuterated and protonated pyruvate exhibited a kinetic isotope effect, showing that the proton transfer is (partly) rate determining and is an essential step in the mechanism of phosphoryl group transfer. Under certain reaction conditions, a more than proportional increase of the 14C exchange rate with increasing total enzyme concentration was observed, indicating that only the dimeric form of enzyme I is phosphorylated. From the dependence of the 14C exchange rate on the phosphoenolpyruvate and pyruvate concentrations, the forward and reverse 2nd-order rate constants of the reaction were determined to be 3 .times. 107 and 8 .times. 105 M-1 min-1, respectively, yielding an equilibrium constant of .apprx. 40 and a .DELTA.G.degree. [Gibbs free energy change] for enzyme I phosphorylation of -2.3 kcal/mol. The significance of the values of these rate constants for the thermodynamics of the phosphotransferase system is discussed.