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Reactivity of 3‐HBA‐6‐Hydroxylase with Diethylpyrocarbonate and N s‐Bromosuccinimide: Effect of Chemical Modifications on Kinetic and Spectral Properties of the Enzyme
Author(s) -
Sumathi Suresh,
Dasgupta Deepak
Publication year - 2000
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp000048g
Subject(s) - chemistry , enzyme , tryptophan , histidine , kinetics , titration , stereochemistry , substrate (aquarium) , enzyme kinetics , residue (chemistry) , n bromosuccinimide , reactivity (psychology) , chemical modification , biochemistry , active site , amino acid , organic chemistry , halogenation , biology , medicine , ecology , physics , alternative medicine , pathology , quantum mechanics
The rapid inactivation of 3‐HBA‐6‐hydroxylase by 100 μM diethylpyrocarbonate or 40 μM 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.