Phosphoenolpyruvate Synthetase

The enzyme phosphoenolpyruvate synthetase from Escherichia coli catalyzes the over-all reaction: ATP + pyruvate ⇌ phosphoenolpyruvate + AMP + Pi. Previous investigations had established the formation of a phosphoenzyme EP as an intermediate and had led to the suggestion that the first step in the reaction was the formation of a pyrophosphoryl enzyme, E + ATP ⇌ EPP + AMP, followed by the hydrolysis of EPP: EPP + H2O ⇌ EP + Pi. Attempts were made to establish this sequence of reactions by (a) the substitution of ATP as substrate with analogues in which the oxygen bridge between the β- and the γ-phosphorus is replaced with carbon or nitrogen and are consequently resistant to hydrolysis at the γ-phosphate and (b) the investigation of the Pi-H218O exchange reaction catalyzed by EP. The 5'-adenylylmethylene diphosphonate analogue was shown to be a competitive inhibitor with respect to ATP in the over-all reaction with a Ki equal to 2.1 µm. It was shown that, unlike ATP, the phosphonate analogue did not lead to the formation of AMP. In addition, preliminary incubation of the enzyme with the phosphonate analogue or with 5'-adenylylimidodiphosphate did not block subsequent phosphorylation of the enzyme by phosphoenolpyruvate, nor did preliminary incubation with the phosphonate analogue block the 14C-pyruvate-phosphoenolpyruvate exchange reaction. The presence of AMP is required for the rate of the H218O-Pi exchange to proceed at a rate comparable to the Pi-ATP exchange, even when the enzyme is maintained in the EP form with ATP or phosphoenolpyruvate. It is concluded that a free pyrophosphoryl enzyme is not an intermediate in the P-enolpyruvate synthetase reaction and a reaction sequence consistent with all of the experimental data is proposed.