Investigations of the partial reactions catalyzed by pyruvate phosphate dikinase.
The kinetic mechanism of pyruvate phosphate dikinase (PPDK) from Bacteroides symbiosus was investigated with several different kinetic diagnostics. Initial velocity patterns were intersecting for AMP/PPi and ATP/Pi substrate pairs and parallel for all other substrate pairs. PPDK was shown to catalyze [14C]pyruvate in equilibrium phosphoenolpyruvate (PEP) exchange in the absence of cosubstrates, [14C]AMP in equilibrium ATP exchange in the presence of Pi/PPi but not in their absence, and [32P]Pi in equilibrium PPi exchange in the presence of ATP/AMP but not in their absence. The enzyme was also shown, by using [alpha beta-18O, beta, beta-18O2]ATP and [beta gamma-18O, gamma, gamma, gamma-18O3]ATP and 31P NMR techniques, to catalyze exchange in ATP between the alpha beta-bridge oxygen and the alpha-P nonbridge oxygen and also between the beta gamma-bridge oxygen and the beta-P nonbridge oxygen. The exchanges were catalyzed by PPDK in the presence of Pi but not in its absence. These results were interpreted to support a bi(ATP,Pi) bi(AMP,PPi) uni(pyruvate) uni(PEP) mechanism. AMP and Pi binding order was examined by carrying out dead-end inhibition studies. The dead-end inhibitor adenosine 5'-monophosphorothioate (AMPS) was found to be competitive vs AMP, noncompetitive vs PPi, and uncompetitive vs PEP. The dead-end inhibitor imidodiphosphate (PNP) was found to be competitive vs PPi, uncompetitive vs AMP, and uncompetitive vs PEP. These results showed that AMP binds before PPi. The ATP and Pi binding order was studied by carrying out inhibition, positional isotope exchange, and alternate substrate studies.(ABSTRACT TRUNCATED AT 250 WORDS)
Wang
H C
HC
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Ciskanik
L
L
Dunaway-Mariano
D
D
von der Saal
W
W
Villafranca
J J
JJ
D000227:Adenine Nucleotides / Q000378:metabolism / Q000494:pharmacology; D001439:Bacteroides / Q000201:enzymology*; D002250:Carbon Radioisotopes; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008433:Mathematics; D010710:Phosphates / Q000494:pharmacology; D010761:Phosphorus Radioisotopes; D010770:Phosphotransferases / Q000378:metabolism*; D011772:Pyruvate, Orthophosphate Dikinase / Q000302:isolation & purification / Q000378:metabolism*
D000227:Adenine Nucleotides; D002250:Carbon Radioisotopes; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D010770:Phosphotransferases; D011772:Pyruvate, Orthophosphate Dikinase