Interaction of 3-hydroxybenzoate-6-hydroxylase with cibacron blue

Abstract

Cibacron blue is a potent inhibitor of 3-HBA-6-hydroxylase at a concentration < 1 μM. Kinetic analyses revealed that at a concentration below 0.5 μM 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 (K_a) and stoichiometry (r) for the enzyme-dye complex.

Keywords::

• Cibacron blue
• circular dichroism
• competitive
• flavin
• fluorescence
• 3-hydroxybenzoate
• inhibition
• uncompetitive
• monooxygenase
• 3-hydroxybenzoate-6-hydroxylase

Acknowledgements

The financial support given by the Indian Institute of Science, Bangalore, India is greatly appreciated. We also acknowledge S. L. Nagraj for his excellent technical assistance.