Comparison of pH-dependent allostery and dissociation for phosphofructokinases from Artemia embryos and rabbit muscle: nature of the enzymes acylated with diethylpyrocarbonate.
Purified Artemia phosphofructokinase (PFK), unlike the rabbit skeletal muscle enzyme, displays allosteric kinetics at pH 8, a feature that is functionally significant since the intracellular pH of the developing brine shrimp embryo is greater than or equal to 7.9. Catalytic activity of the Artemia enzyme is severely suppressed by acidic pH even when assayed at the adenylate nucleotide concentrations existing in anaerobic embryos, which is consistent with the lack of a Pasteur effect in these organisms. For both PFK homologs, carbethoxylation reduces the sensitivity to ATP and citrate inhibition, the cooperativity as a function of fructose 6-phosphate concentration and the degree of activation in the presence ADP, AMP, and fructose 2,6-bisphosphate. Considering the role of histidine protonation in PFK allosteric control, the capacity for regulatory kinetics seen at pH 8 in the Artemia enzyme could be explained in part by upward shifts in pKa values of ionizable residues. pH-induced dissociation of tetrameric Artemia PFK into inactive subunits does not occur during catalytic inhibition at acidic pH (pH 6.5, 6 degrees C), as judged by 90 degree light scattering. This observation contrasts markedly with the dimerization and inactivation of rabbit PFK, but is shown not to be unique when compared to other selected PFK homologs. Neither the acute pH sensitivity of Artemia PFK nor the pH-induced hysteretic inactivation displayed by the rabbit enzyme are altered by carbethoxylation, suggesting that ionizable residues involved in these two processes are not the same ones involved in allosteric kinetics.
D000215:Acylation; D000227:Adenine Nucleotides / Q000494:pharmacology; D000494:Allosteric Regulation; D000818:Animals; D001156:Artemia / Q000196:embryology; D004047:Diethyl Pyrocarbonate / Q000378:metabolism*; D004789:Enzyme Activation; D005561:Formates / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits
D000227:Adenine Nucleotides; D005561:Formates; D046911:Macromolecular Substances; D010732:Phosphofructokinase-1; D004047:Diethyl Pyrocarbonate