title: Chemical and kinetic evidence for an essential histidine in horseradish peroxidase for iodide oxidation.
abstract: Horseradish peroxidase (HRP), when incubated with diethylpyrocarbonate (DEPC), shows a time-dependent loss of iodide oxidation activity. The inactivation follows pseudo-first order kinetics with a second order rate constant of 0.43 min-1 M-1 at 30 degrees C and is reversed by neutralized hydroxylamine. The difference absorption spectrum of the modified versus native enzyme shows a peak at 244 nm, characteristic of N-carbethoxyhistidine, which is diminished by treatment with hydroxylamine. Correlation between the stoichiometry of histidine modification and the extent of inactivation indicates that out of 2 histidine residues modified, one is responsible for inactivation. A plot of the log of the reciprocal half-time of inactivation against log DEPC concentration further suggests that only 1 histidine is involved in catalysis. The rate of inactivation shows a pH dependence with an inflection point at 6.2, indicating histidine derivatization by DEPC. Inactivation due to modification of tyrosine, lysine, or cysteine has been excluded. CD studies reveal no significant change in the protein or heme conformation following DEPC modification. We suggest that a unique histidine residue is required for maximal catalytic activity of HRP for iodide oxidation.
authors.affiliation: Department of Physiology, Indian Institute of Chemical Biology, Calcutta.
authors.forename: D K
authors.initials: DK
authors.lastname: Bhattacharyya
authors.forename: U
authors.initials: U
authors.lastname: Bandyopadhyay
authors.forename: R K
authors.initials: RK
authors.lastname: Banerjee
mesh_terms: D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D006639:Histidine; D006735:Horseradish Peroxidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007454:Iodides / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D013056:Spectrophotometry, Ultraviolet
chemical_list: D007454:Iodides; D006639:Histidine; D006735:Horseradish Peroxidase; D004047:Diethyl Pyrocarbonate