Studies of the structure–function relationships of Neurospora crassa pyruvate kinase: interaction with blue dextran – Sepharose and Cibacron blue 3G-A

Abstract

Blue dextran - Sepharose and Cibacron blue 3G-A interact with pyruvate kinase of N. spora crassa. The enzyme is readily released from the substituted Sepharose column by elution with 0.17 M potassium phosphate buffer (pH 7.9), or 2 mM fructose 1,6-diphosphate (FDP), but not with either of the substrates, ADP and phosphoenolpyruvate (PEP), at 2 mM. Cibacron blue 3G-A is a noncompetitive inhibitor of pyruvate kinase with respect to both substrates. It appears to compete with the allosteric effector, FDP, for binding to the enzyme surface. A lack of elution of the enzyme from the immobilized blue dextran matrix by adenine nucleotides and the absence of a difference spectrum in the 650-700 nm range suggest that a dinucleotide-fold substructure is not implicated in the dye binding sites on pyruvate kinase. The interaction of Cibacron blue 3G-A and this enzyme can be followed fluorometrically; incremental addition of the dye to the enzyme solution results in a progressive decrease in the fluorescence of surface tryptophanyl residues. The quenching of fluorescence of exposed aromatic groups is subject to reversal following addition of FDP to the pyruvate kinase, Cibacron blue complex.