Modulation of phosphofructokinase behavior by chemical modifications during the immobilization process.
Phosphofructokinase was immobilized within a protein membrane or on soluble protein polymers using glutaraldehyde as cross-linking reagent. The native enzyme was also modified chemically, using the cross-linking reagent alone. A comparative kinetic investigation of these preparations was carried out. The catalytic activity of the chemically modified enzyme and its affinity towards fructose 6-phosphate decreased significantly; the modified enzyme lost its cooperative properties and the allosteric regulation by AMP was affected. When the chemical treatment was performed in the presence of effectors (AMP or ATP) the allosteric transition induced by AMP was restored, suggesting that the cross-linking reagent modified the AMP regulatory sites, albeit no higher-substrate-affinity enzyme conformation was frozen. Molecular data showed that glutaraldehyde produced intramolecular then intermolecular bonds as its concentration increased. When the enzyme was immobilized into protein membranes or on soluble polymers, the enzyme behavior was quite similar: decrease of affinity towards fructose 6-phosphate but no changes in cooperative properties and modifications of allosteric transition induced by AMP. When AMP was present during the immobilisation process, the enzyme immobilized in this way was no longer sensitive to effectors, either AMP or ATP. It showed Michaelian behavior and higher substrate affinity quite similar to that of the native enzyme. The data suggested that a higher-substrate-affinity enzymatic form was most probably stabilized by immobilization.
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000495:Allosteric Site / Q000187:drug effects; D055598:Chemical Phenomena; D002621:Chemistry; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005976:Glutaral / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012710:Serum Albumin, Bovine
D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D046911:Macromolecular Substances; D012710:Serum Albumin, Bovine; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; D005976:Glutaral