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Mechanism of Inhibition of Horseradish Peroxidase-catalysed Iodide Oxidation by EDTA

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Title Mechanism of Inhibition of Horseradish Peroxidase-catalysed Iodide Oxidation by EDTA
 
Creator Bhattacharya, Dipak Kr
Adak, Subrata
Bandyopadhyay, Uday
Banerjee, Ranajit K
 
Subject Structural Biology & Bioinformatics
 
Description EDTA inhibits horseradish peroxidase (HRP)-catalysed iodide
oxidation in a concentration and pH-dependent manner. It is
more effective at pH 6 than at lower pH values. A plot of log
K values as a function of pH yields a sigmoidal curve from
which a pKa value of 5.4 can be calculated for an ionizable group
on the catalytically active HRP for EDTA inhibition. Among the
structural analogues of EDTA, tetramethylethylenediamine
(TEMED) is 80 % as effective as EDTA, whereas the EDTA-Zn2+
chelate and EGTA are ineffective. Kinetic studies indicate that
EDTA competitively inhibits iodide oxidation. Spectral studies
show that EDTA can quickly reduce compound I to compound
II, but reduction of preformed compound II to the native enzyme
is relatively slow, as demonstrated by the time-dependent spectral
shift from 417 nm to 402 nm through an isosbestic point at
408 nm. Under steady-state conditions, in a reaction mixture
containing HRP, EDTA and H202, the enzyme remains in the
compound-II form, with absorption maxima at 417, 527 and
556 nm. Direct evidence for one-electron oxidation of EDTA by
HRP intermediates is provided by the appearance of an e.s.r.
signal of a 5,5-dimethyl-1-pyrroline N-oxide (spin trap)-EDTA
radical adduct [aN (hyperfine splitting constant) = 1.5 mT] in
e.s.r. studies. The signal intensity, however, decreases in the
presence of iodide. The KD of the HRP-EDTA complex obtained
from optical difference spectroscopy increases with an increase in
iodide concentration, and the double-reciprocal plot for EDTA
binding indicates that EDTA and iodide compete for the same
binding site for oxidation. We suggest that EDTA inhibits iodide
oxidation by acting as an electron donor.
 
Date 1994
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/1033/1/biojdipak.pdf
Bhattacharya, Dipak Kr and Adak, Subrata and Bandyopadhyay, Uday and Banerjee, Ranajit K (1994) Mechanism of Inhibition of Horseradish Peroxidase-catalysed Iodide Oxidation by EDTA. Biochemical Journal, 298. pp. 281-288.
 
Relation http://dx.doi.org/
http://www.eprints.iicb.res.in/1033/