Interaction of 3-hydroxybenzoate-6-hydroxylase with cibacron blue.
Cibacron blue is a potent inhibitor of 3-HBA-6-hydroxylase at a concentration < 1 microM. Kinetic analyses revealed that at a concentration below 0.5 microM the dye behaves as an uncompetitive inhibitor with respect to 3-HBA and competes with NADH for the same site on the enzyme. The alteration of the near-UV CD spectrum and quenching of the emission fluorescence of the enzyme by cibacron blue indicates a significant alteration in the environment of aromatic amino acid residues due to a stacking interaction and subtle conformatiodnal changes in the enzyme. The concentration-dependent quenching of the intrinsic fluorescence of the enzyme by cibacron blue was employed to determine the binding parameters such as association constant (Ka) and stoichiometry (r) for the enzyme-dye complex.
Sumathi
S
S
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India. sumathis@iitb.ac.in
Dasgupta
Dipak
D
D001665:Binding Sites; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014227:Triazines / Q000378:metabolism*
D004791:Enzyme Inhibitors; D014227:Triazines; D009243:NAD; C012842:Cibacron Blue F 3GA; D006899:Mixed Function Oxygenases; C023315:3-hydroxybenzoate-6-hydroxylase