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Mechanism of horseradish peroxidase inactivation by benzhydrazide: a critical evaluation of arylhydrazides as peroxidase inhibitors.

ABSTRACT: Many compounds are oxidized by haem enzymes, such as peroxidases and cytochromes P450, to highly reactive intermediates that function as enzyme inactivators. To evaluate the potential of arylhydrazides as selective metabolically activated peroxidase inhibitors, the mechanism of HRPC (horseradish peroxidase isoenzyme C) inhibition by BZH (benzhydrazide) was investigated in detail. No oxygen consumption was detected in BZH solutions at pH 7.0-12.0, but addition of HRPC resulted in significant O2 uptake above pH 8.0, indicating that the enzyme catalyses BZH oxidation. Addition of H2O2 to HRPC plus BZH activates the latter as an inhibitor. This involves the three-electron oxidation of BZH in one-electron steps by the peroxidase catalytic intermediates, Compounds I and II, to produce a benzoyl radical that covalently alters the active site and inhibits peroxidase activity. Alternatively, the benzoyl radical could be produced by di-imide (NH=NH) elimination from the BZH radical. Production of Compound III (oxyperoxidase) followed by p-670 (m/z =583, biliverdin-like derivative) was observed for HRPC incubated with excess H2O2, and the addition of BZH resulted in an increase in the rate of p-670 production. BZH is an inefficient inhibitor of HRPC with a K(I) of 80 muM, an apparent inactivation rate constant (k(inact)) of 0.035 min(-1), and an IC50 of 1.0 mM. This prompted the investigation of HRPC inactivation by a series of related arylhydrazides with known binding affinities for HRPC. The hydrazide with the highest affinity (2-naphthoichydrazide; K(d)=5.2 muM) was also found to be the most effective inhibitor with K(I), k(inact) and IC50 values of 14 muM, 0.14 min(-1) and 35 muM, respectively.

SUBMITTER: Aitken SM

PROVIDER: S-EPMC1223706 | biostudies-other | 2003 Nov

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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Mechanism of horseradish peroxidase inactivation by benzhydrazide: a critical evaluation of arylhydrazides as peroxidase inhibitors.

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