Isolation of a pyrophosphoryl form of pyruvate, phosphate dikinase from Propionibacteria.

Pyruvate, phosphate dikinase from Propionibacterium shermanii catalyzes the formation of P-enolpyruvate, AMP, and inorganic pyrophosphate from pyruvate, ATP, and orthophosphate; the mechanism involves three partial reactions and three forms of the enzyme: pyrophosphoryl-enzyme, phosphoryl-enzyme, and free enzyme. The phosphoryl-enzyme was prepared by incubation with P-enolpyruvate and isolated by gelchromatography. The phosphoryl-enzyme was converted to (32)P(31)P-enzyme and [(32)P]Pi by incubation with [(32)P]PPi; 1 mol of pyrophosphoryl-enzyme was formed per mol of enzyme of molecular weight 150,000. The labeled enzyme released its radioactivity upon incubation with Pi or AMP to produce the expected [(33)P]PPi or [gamma-(32)P]ATP, respectively. Hydrolysis of the pyrophosphoryl-enzyme with dilute acid yielded PPi. The beta,gamma-methylene analogue of ATP was reactive in exchange reactions with [(14)C]AMP. To our knowledge, this is the first proven example of a pyrophosphoryl-enzyme.