Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: mechanism of the phosphoryl-group transfer from phosphoenolpyruvate to HPr

Abstract

The mechanism of phosphoryl-group transfer from phosphoenolpyruvate (PEP) to HPr [heat-stable protein cofactor for bacterial enzymes], catalyzed by enzyme I of the E. coli PEP-dependent phosphotranferase system, was studied in vitro. Steady-state kinetics and isotope exchange measurements revealed that this reaction cannot be described by a classical ping-pong mechanism although phosphoenzyme I acts as an intermediate. The kinetic data indicate that HPr and PHPr occupy binding sites on enzyme I that do not overlap with the binding sites for PEP and pyruvate. As a result, binding interactions between HPr and enzyme I exist regardless of their phosphorylated state. A general mechanism is presented that describes the phosphorylation of HPr. The physiological implications of this mechanism are discussed.