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Reactivity of 3-HBA-6-hydroxylase with diethylpyrocarbonate and N-bromosuccinimide: effect of chemical modifications on kinetic and spectral properties of the enzyme.

Authors
  • Sumathi, S
  • Dasgupta, D
Type
Published Article
Journal
Biotechnology Progress
Publisher
Wiley (John Wiley & Sons)
Publication Date
Jan 01, 2000
Volume
16
Issue
4
Pages
577–582
Identifiers
PMID: 10933831
Source
Medline
License
Unknown

Abstract

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.

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