Phosphoenolpyruvate carboxylase of Escherichia coli. Studies on multiple conformational states elicited by allosteric effectors with a fluorescent probe, 1-anilinonaphthalene-8-sulfonate.
Conformational change of phosphoenolpyruvate carboxylase (orthophosphate: oxaloacetate carboxy-lyase (phosphorylating), EC 4.1.1.31) induced by allosteric effectors was investigated using a hydrophobic probe, 1-anilinonaphthalene-8-sulfonate (ANS). Kinetic experiments suggested that ANS binds with the enzyme at the sites which are not involved in the catalytic and regulatory functions, though it partially inhibits the enzyme activity with half-saturation concentration (S0.5) of 38.5 muM. Binding experiments showed that a maximum of 2 mol of ANS are able to bind with 1 mol of the enzyme subunit presumably with an equal dissociation constant to each other (34.5 muM). Flourescence emission of ANS was markedly increased by binding with the enzyme. L-Aspartate, the allosteric inhibitor, and CoASAc and fructose 1,6-bisphosphate (Fru-1,6-P2) the allosteric activators, produced various degrees of change in fluorescence, when added singly or in combinations. The changes were shown to be attributable to the allosteric interactions between the enzyme and effectors from some criteria such as structural specificity, half-saturation concentrations, and heterotropic-homotropic interactions of the ligands. It was concluded from these analyses that the enzyme can be in at least four conformational states which are distinct from each other. Especially noteworthy is the finding that the enzyme, upon simultaneous binding of CoASAc and Fru-1,6-P2, takes a new conformation which is enterely different from those induced by sole binding of each effector. In addition, the heterotropic interaction between the activator and the inhibitor was observed through conformational change by the ANS method, as observed in the kinetic studies.
D000105:Acetyl Coenzyme A / Q000494:pharmacology; D000494:Allosteric Regulation; D000495:Allosteric Site; D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D001224:Aspartic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005636:Fructosephosphates / Q000494:pharmacology; D006598:Hexosediphosphates / Q000494:pharmacology; D007700:Kinetics; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D005636:Fructosephosphates; D006598:Hexosediphosphates; D001224:Aspartic Acid; D000105:Acetyl Coenzyme A; D010729:Phosphoenolpyruvate Carboxykinase (GTP)