Pyruvate, phosphate dikinase of Bacteroides symbiosus. Catalysis of partial reactions and formation of phosphoryl and pyrophosphoryl forms of the enzyme.

Pyruvate, phosphate dikinase from Bacteroides symbiosus catalyzes the conversion of pyruvate, ATP, and Pi to Penolpyruvate, AMP, and PP,. Initial velocity studies by both the forward and reverse reactions in which the three substrates were varied in a constant ratio were found to be in accord with a Tri Uni Uni Ping Pong mechanism. The reaction involves three partial reactions which were investigated by the following exchanges: (a) [WIAMP =F ATP, (b) [=PlP , = PP,, and (c) [Wlpyruvate % P-enolpyruvate. Exchanges a and c were independent of other substrates but b was dependent on the presence of either P-enolpyruvate or ATP. These results also are in accord with the Tri Uni Uni Ping Pong mechanism. Phosphoryl enzyme was isolated by incubation of the enzyme with 32P-enolpyruvate and pyrophosphoryl enzyme by incubation with either Penolpyruvate and [Y’PIPPi or with [/3or yJ2PlATP. In contrast, incubation of the enzyme with W-labeled substrates did not label the enzyme, showing that only the phosphoryl moieties are transferred to the enzyme. The covalent nature of the phosphorylated intermediates was shown by acrylamide gel electrophoresis in 6.0 M urea in which the 3’LP counts remained associated with the protein bands. On acid hydrolysis, phosphoric and pyrophosphoric acid were obtained from the phosphoryl and pyrophosphoryl enzyme, respectively. The pyrophosphoryl enzyme is less stable than the phosphoryl enzyme and there was low recovery of pyrophosphoric acid from this form of the enzyme. Titration of the enzyme with either 32P-enolpyruvate or F”PlPP,, showed that -2 mol of phosphoryl or pyrophosphoryl groups are bound per mol of enzyme of 150,000 daltons. Loss of enzymatic activity occurs during storage but this was not accompanied by a corresponding decrease of phosphorylation of the enzyme. The equilibrium constant for hydrolysis of the phosphoryl enzyme was estimated using 32P and P-enol[Wlpyruvate. The average K,, was found (at 25”, pH 7.2) to be 0.24 from which the free energy of hydrolysis of the phosphoryl enzyme was found to be about -14 kcal/mol.