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

The gradual quenching of the emission fluorescence of 3-HBA in the visible region upon titration with 3-HBA-6-hydroxylase and distinct changes in the near-UV circular dichroic spectrum of the enzyme in the presence of substrate suggest the formation of a stable enzyme-substrate complex. The binding of aromatic substrate 3-hydroxybenzoate to 3-hydroxybenzoate-6-hydroxylase occurs without gross changes in the backbone structure of the enzyme. The binding strength of the ES complex is partially reduced upon chemical modification of arginine, histidine, or tryptophan residues of enzyme, probably implicating their concerted action in the binding of substrate to enzyme. Partial inactivation of enzyme and diminished stability of the ES complex in response to treatment with 1 M urea could be ascribed to localized effects of the denaturant.