Isolation of a Pyrophosphoryl Form of Pyruvate, Phosphate Dikinase from Propionibacteria*

AUTOR(ES)

Milner, Yoram

RESUMO

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 32P31P-enzyme and [32P]Pi by incubation with [32P]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 [33P]PPi or [γ-32P]ATP, respectively. Hydrolysis of the pyrophosphoryl-enzyme with dilute acid yielded PPi. The β,γ-methylene analogue of ATP was reactive in exchange reactions with [14C]AMP. To our knowledge, this is the first proven example of a pyrophosphoryl-enzyme.

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