Reactivity of 3‐HBA‐6‐Hydroxylase with Diethylpyrocarbonate and Ns‐Bromosuccinimide: Effect of Chemical Modifications on Kinetic and Spectral Properties of the Enzyme

Abstract

AbstractThe 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.