Studies of the structure--function relationships of Neurospora crassa pyruvate kinase: interaction with blue dextran--sepharose and Cibacron blue 3G-A.
Blue dextran--Sepharose and Cibacron blue 3G-A interact with pyruvate kinase of Neurospora 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- to 700-nm range suggest that a "dinucleotide-fold" substructure is not implicated in the dye binding sites on pyruvate kiase. The interaction of Cibacron blue 3G-A and this enzyme can be followed fluorometrically; incremental additon 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 pyruvte kinase--Cibacron blue complex.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000873:Anthracenes / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D005635:Fructosediphosphates / Q000494:pharmacology; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D011770:Pyruvate Kinase / Q000378:metabolism*; D012685:Sepharose; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014227:Triazines
D000873:Anthracenes; D005635:Fructosediphosphates; D014227:Triazines; C012842:Cibacron Blue F 3GA; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D012685:Sepharose; D011770:Pyruvate Kinase