Biochemical, structural, and kinetic characterization of PPi‐dependent phosphoenolpyruvate carboxykinase from Propionibacterium freudenreichii

Abstract

AbstractPhosphoenolpyruvate carboxykinases (PEPCK) are a well‐studied family of enzymes responsible for the regulation of TCA cycle flux, where they catalyze the interconversion of oxaloacetic acid (OAA) and phosphoenolpyruvate (PEP) using a phosphoryl donor/acceptor. These enzymes have typically been divided into two nucleotide‐dependent classes, those that use ATP and those that use GTP. In the 1960's and early 1970's, a group of papers detailed biochemical properties of an enzyme named phosphoenolpyruvate carboxytransphosphorylase (later identified as a third PEPCK) from Propionibacterium freudenreichii (PPi‐PfPEPCK), which instead of using a nucleotide, utilized PPi to catalyze the same interconversion of OAA and PEP. The presented work expands upon the initial biochemical experiments for PPi‐PfPEPCK and interprets these data considering both the current understanding of nucleotide‐dependent PEPCKs and is supplemented with a new crystal structure of PPi‐PfPEPCK in complex with malate at a putative allosteric site. Most interesting, the data are consistent with PPi‐PfPEPCK being a Fe2+ activated enzyme in contrast with the Mn2+ activated nucleotide‐dependent enzymes which in part results in some unique kinetic properties for the enzyme when compared to the more widely distributed GTP‐ and ATP‐dependent enzymes.