Reactivity of 3-HBA-6-hydroxylase with diethylpyrocarbonate and N-bromosuccinimide: effect of chemical modifications on kinetic and spectral properties of the enzyme.
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.
Sumathi
S
S
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
Dasgupta
D
D
D001974:Bromosuccinimide / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000378:metabolism*; D007202:Indicators and Reagents; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D013057:Spectrum Analysis
D007202:Indicators and Reagents; D006899:Mixed Function Oxygenases; C023315:3-hydroxybenzoate-6-hydroxylase; D001974:Bromosuccinimide; D004047:Diethyl Pyrocarbonate