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Iodide modulation of the EDTA-induced iodine reductase activity of horseradish peroxidase by interaction at or near the EDTA-binding site.

ABSTRACT: Horseradish peroxidase (HRP) catalyses the reduction of iodinium ion (I+) to iodide by H2O2 in the presence of EDTA. I+ reduction occurs optimally at pH 6 whereas the enzyme catalyses iodide oxidation optimally at pH 3.5. Thus the two activities reside on the same enzyme with two characteristic pH optima. Iodide modulates the expression of the reductase activity by EDTA. Higher concentrations of iodide inhibit the reductase activity by EDTA. Nitrite, an electron donor, acts similarly to iodide. Both EDTA and nitrite competitively inhibit iodide oxidation, indicating that they compete with iodide for the same binding site for electron flow to the haem iron group. However, unlike iodide, EDTA converts compound I, not into the native enzyme, but into a compound absorbing at 416 nm which reduces I+ and then returns to the native form. The apparent equilibrium dissociation constant, KD, for the formation of the EDTA-HRP complex (15 mM) is doubled in the presence of iodide, indicating interference with EDTA binding by iodide. EDTA binds away from the haem iron centre and not through intramolecular Ca2+. The pH-dependence of EDTA binding indicates that an ionizable group of the enzyme with pKa 5.8, presumably a distal histidine, controls the binding. The data suggest that iodide competes with EDTA for compound I and modulates the iodine reductase activity by limiting the formation of the 416 nm-absorbing active compound.

SUBMITTER: Bhattacharyya DK

PROVIDER: S-EPMC1132207 | biostudies-other | 1993 Jan

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: Horseradish peroxidases (HRPs) from Armoracia rusticana have long been utilized as reporters in various diagnostic assays and histochemical stainings. Regardless of their increasing importance in the field of life sciences and suggested uses in medical applications, chemical synthesis and other industrial applications, the HRP isoenzymes, their substrate specificities and enzymatic properties are poorly characterized. Due to lacking sequence information of natural isoenzymes and the low levels of HRP expression in heterologous hosts, commercially available HRP is still extracted as a mixture of isoenzymes from the roots of A. rusticana. RESULTS: In this study, a normalized, size-selected A. rusticana transcriptome library was sequenced using 454 Titanium technology. The resulting reads were assembled into 14871 isotigs with an average length of 1133 bp. Sequence databases, ORF finding and ORF characterization were utilized to identify peroxidase genes from the 14871 isotigs generated by de novo assembly. The sequences were manually reviewed and verified with Sanger sequencing of PCR amplified genomic fragments, resulting in the discovery of 28 secretory peroxidases, 23 of them previously unknown. A total of 22 isoenzymes including allelic variants were successfully expressed in Pichia pastoris and showed peroxidase activity with at least one of the substrates tested, thus enabling their development into commercial pure isoenzymes. CONCLUSIONS: This study demonstrates that transcriptome sequencing combined with sequence motif search is a powerful concept for the discovery and quick supply of new enzymes and isoenzymes from any plant or other eukaryotic organisms. Identification and manual verification of the sequences of 28 HRP isoenzymes do not only contribute a set of peroxidases for industrial, biological and biomedical applications, but also provide valuable information on the reliability of the approach in identifying and characterizing a large group of isoenzymes.

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Iodide modulation of the EDTA-induced iodine reductase activity of horseradish peroxidase by interaction at or near the EDTA-binding site.

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