Mechanism of inhibition of horseradish peroxidase-catalysed iodide oxidation by EDTA

Abstract

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 Kiapp. 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 H2O2, 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.

Molecular Analysis of a 4-Dimethylallyltryptophan Synthase from Malbranchea aurantiaca

“…In a H101N variant of human liver arginase, EDTA exhibited a predominant competitive inhibition, and its carboxylic acid group was presumably important for this effect (34). EDTA was also shown to be a competitive inhibitor of horseradish peroxidase by acting as an electron donor to compete with substrate iodine (33). Moreover, Chan and Anthony (39) found that EDTA bound to lysyl or arginyl residues near the cytochromebinding domain of methanol dehydrogenase.…”

“…3B), demonstrating EDTA as a reversible inhibitor of MaPT. Interestingly, 5 mM EDTA was also reported to reduce CloQ prenyltransferase activity to 25% (23), and inhibits horseradish peroxidase, human liver arginase, and lignin peroxidase H2 in a metal ion-independent manner (32)(33)(34).…”

Non-chelating inhibition of the H101N variant of human liver arginase by EDTA

“…As in a few other examples [30][31][32], we conclude here that low concentrations of EDTA inhibit the H101N variant of human liver arginase independently of Mn 2þ chelation. This is important because it means that activities measured in the presence of EDTA cannot be taken as prima facie evidence for the essentiality of a metal ion for arginase activity.…”

An improved method for bright-field imaging of arbuscular mycorrhizal fungi in plant roots

“…Plant background signal was significantly suppressed by the addition of bovine serum albumin. The HRP reaction was stopped by the addition of EDTA, which inhibits DAB oxidation by HRP (Bhattacharyya et al 1994). Precipitates in roots soaked in TE buffer were stable for at least 2 months at 4°C.…”