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] [from E. coli] induced by allosteric effectors was investigated using a hydrophobic probe, 1-anilino-naphthalene-8-sulfonate (ANS). Kinetic experiments suggested that ANS binds with the enzyme at sites which are not involved in catalytic and regulatory functions, though it partially inhibits the enzyme activity with half-saturation concentration (S0.5) of 38.5 .mu.M. Binding experiments showed that a maximum of 2 mol of ANS can bind with 1 mol of the enzyme subunit presumably with an equal Kd to each other (34.5 .mu.M). Fluorescence emission of ANS was markedly increased by binding with the enzyme. L-Aspartate, the allosteric inhibitor, CoASAc [acetyl CoA] and fructose 1,6-biphosphate (Fru-1,6-P2), the allosteric activators, produced various degrees of change in fluorescence when added singly or in combinations. The changes were attributable to allosteric interactions between the enzyme and effectors as shown by structural specificity, half-saturation concentrations and heterotropic-homotropic interactions of ligands. The enzyme apparently can be in at least 4 conformational states which are distinct from each other. The enzyme, upon simultaneous binding of CoASAc and Fru-1,6-P2, takes a new conformation which is entirely different from those induced by sole binding of each effector. The heterotropic interaction between the activator and the inhibitor was observed through conformational change by the ANS method, in the kinetic studies.