The rapid inactivation of 3-HBA-6-hydroxylase by 100 microM diethylpyrocarbonate or 40 microM N-bromosuccinimide and protection offered by the substrate, 3-hydroxybenzoate, against these chemical modifications implicate the involvement of histidine and tryptophan in the catalytic activity of the enzyme. Inactivation of the enzyme by diethylpyrocarbonate followed pseudo-first-order kinetics, and an "n" value of 1.3 was obtained. Inactivation of the enzyme by N-bromosuccinimide was instantaneous and failed to follow pseudo-first-order kinetics. Distinct and incremental changes in the UV absorption, emission fluorescence, and near UV-CD spectra of the enzyme upon its titration with increasing concentrations of diethylpyrocarbonate or N-bromosuccinimide may be ascribed to modification and/or changes in the microenvironment of aromatic amino acid residue(s) such as tryptophan in the enzyme.
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.
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.
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