Kinetic mechanism and metabolic role of pyruvate phosphate dikinase from Entamoeba histolytica.
The kinetic mechanism and the metabolic role of pyruvate phosphate dikinase from Entamoeba histolytica were investigated. The initial velocity patterns in double reciprocal plots were parallel for the phosphoenolpyruvate/AMP and phosphoenolpyruvate/pyrophosphate substrate pairs and intersecting for the AMP/pyrophosphate pair. This suggests a kinetic mechanism with two independent reactions. The rate of ATP synthesis at saturating and equimolar concentrations of phosphoenolpyruvate, AMP, and pyrophosphate was inhibited by phosphate, which is consistent with an ordered steady-state mechanism. Enzyme phosphorylation by [(32)P(i)]pyrophosphate depends on the formation of a ternary complex between AMP, pyrophosphate, and pyruvate phosphate dikinase. In consequence, the reaction that involves the AMP/pyrophosphate pair follows a sequential steady-state mechanism. The product inhibition patterns of ATP and phosphate versus phosphoenolpyruvate were noncompetitive and uncompetitive, respectively, suggesting that these products were released in an ordered process (phosphate before ATP). The ordered release of phosphate and ATP and the noncompetitive inhibition patterns of pyruvate versus AMP and versus pyrophosphate also supported the sequential kinetic mechanism between AMP and pyrophosphate. Taken together, our data provide evidence for a uni uni bi bi pingpong mechanism for recombinant pyruvate phosphate dikinase from E. histolytica. The Delta G value for the reaction catalyzed by pyruvate phosphate dikinase (+2.7 kcal/mol) determined under near physiological conditions indicates that the synthesis of ATP is not thermodynamically favorable in trophozoites of E. histolytica.
Varela-Gómez
Marcela
M
Departamento de Bioquímica, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70242, 04510 México D. F. México.
Moreno-Sánchez
Rafael
R
Pardo
Juan Pablo
JP
Perez-Montfort
Ruy
R
D000249:Adenosine Monophosphate / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000818:Animals; D011756:Diphosphates / Q000378:metabolism / Q000494:pharmacology; D004748:Entamoeba histolytica / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D007700:Kinetics; D010710:Phosphates / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism / Q000494:pharmacology; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011772:Pyruvate, Orthophosphate Dikinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D011756:Diphosphates; D004791:Enzyme Inhibitors; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D010728:Phosphoenolpyruvate; D000255:Adenosine Triphosphate; D011772:Pyruvate, Orthophosphate Dikinase