Kinetic evidence for separate site catalysis by pyruvate phosphate dikinase

Pyruvate phosphate dikinase from Clostridium symbiosum catalyzes the interconversion of adenosine-5'-triphosphate (ATP), orthophosphate (Pi), and pyruvate with adenosine 5'-monophosphate (AMP), inorganic pyrophosphate (PPi), and phosphoenolpyruvate (PEP) using a bi (ATP, Pi) bi (AMP, PPi) uni (pyruvate) uni (PEP) kinetic mechanism and pyrophosphorylenzyme (E-PP) and phosphorylenzyme (E-P) covalent intermediates. The present studies were carried out to determine whether or not the site of catalysis of the E + ATP + P1 E-P + AMP + PPi partial reaction overlaps with that of the E-P + pyruvate E + PEP partial reaction. Single-turnover experiments were carried out to test the effect of binding site occupancy on catalysis at a second site. Saturation of the enzyme with adenyl imidodiphosphate (AMPPNP) inhibited [32P]E-P formation from [beta-32P]ATP and Pi but did not significantly inhibit [32P]E-P formation from [32P]PEP. Likewise, saturation of E-P with AMP did not significantly inhibit [14C]PEP formation from [14C]pyruvate, suggesting separate, largely independent ATP/AMP vs pyruvate/PEP sites. Movement of the phosphorylhistidine residue between reaction sites was probed by testing oxalate as an inhibitor of phosphoryl transfer from E-P to [14C]pyruvate or to [14C]AMP. Both phosphoryl transfers were inhibited. These results were interpreted as evidence for the requirement for phosphorylhistidine release from the pyruvate site prior to participation in catalysis at the nucleotide site.