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.
Thrall
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Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Dunaway-Mariano
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D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D003013:Clostridium / Q000201:enzymology; D007700:Kinetics; D010710:Phosphates / Q000378:metabolism; D011772:Pyruvate, Orthophosphate Dikinase / Q000378:metabolism*; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid
D010710:Phosphates; D011773:Pyruvates; D000249:Adenosine Monophosphate; D019289:Pyruvic Acid; D000255:Adenosine Triphosphate; D011772:Pyruvate, Orthophosphate Dikinase