Roles of Efficient Substrates in Enhancement of Peroxidase-Catalyzed Oxidations

Abstract

Efficient peroxidase substrates may have a critical role in the oxidation of secondary compounds by peroxidases. Hydrazines are often oxidized slowly by peroxidases due, in part, to hydrazine-dependent inactivation of these enzymes. Peroxidase-catalyzed oxidation of hydrazines may be dramatically affected by an efficient peroxidase substrate. We investigated this hypothesis in a model system using the well-known peroxidase substrate chlorpromazine (CPZ) and the hydrazine derivative isoniazid. CPZ stimulated isoniazid oxidation as measured by nitroblue tetrazolium (NBT) reduction and O2 consumption. The kinetics of isoniazid and CPZ oxidation by horseradish peroxidase (HRP) in the presence of both compounds suggested CPZ was acting as an electron transfer mediator between HRP and isoniazid. Indeed, CPZ.+, the product of CPZ oxidation by HRP, was able to oxidize isoniazid. The rate constant for this pH-dependent reaction was (2.6 +/- 0.1) x 10(4) M-1 s-1 at pH 4.5. In the absence of CPZ, isoniazid-dependent irreversible inactivation of HRP was observed. The inactivation process involved the formation of compound III followed by accumulation of irreversibly inactivated HRP. CPZ completely inhibited inactivation. Thus, by acting as a redox mediator and preventing HRP inactivation, CPZ stimulated isoniazid oxidation by several orders of magnitude. Similarly, other efficient peroxidase substrates, such as phenol and tyrosine, were also able to dramatically stimulate isoniazid oxidation by HRP. We suggest that the presence of efficient peroxidase substrates may potentiate the activation of isoniazid and other hydrazines. As such, these substrates may have a vital role in the pharmacological and toxicological properties of hydrazines and other compounds.

Horseradish Peroxidase-Catalyzed Oxidation of Rifampicin: Reaction Rate Enhancement by Co-oxidation with Anti-inflammatory Drugs

“…It has been reported that the catalytic efficiency for oxidation of some peroxidase substrates can be enhanced by the presence of a secondary substance (co-oxidation). 18,19) In confirmation of this, here we verified that the rate of rifampicin oxidation increased in a concentration-dependent manner when TMB was added to the reaction mixture (Fig. 3A).…”

“…For instance, hydroquinone was described as an effective activator of ascorbic acid oxidation, and chlorpromazine was found to promote the oxidation of isoniazid, in both cases by HRP-catalyzed reactions. 18,19) In such cases, the better substrate is oxidized first by the oxidized intermediate forms of peroxidases and its product is the oxidizing agent for the second substrate. This suggests that the extreme differences observed in our results between the anti-inflammatories might be related to their reactivity with HRP.…”

Mechanism-Based Inhibition of Myeloperoxidase by Hydrogen Peroxide: Enhancement of Inactivation Rate by Organic Donor Substrates

“…These reactions might account for the increase in K i for H 2 O 2 due to the hydrazides. Goodwin et al [11] reported that ISD enhanced the H 2 O 2 -dependent inactivation of horseradish peroxidase. The report suggested that inactivation rate enhancement by ISD may involve a mechanism similar to that due to H 2 O 2 alone.…”

“…In this study, we have used a steady state kinetics approach to demonstrate that MPO is irreversibly inactivated by excess H 2 O 2 in the absence of reducing substrate. The rate of the inactivation process is enhanced in the presence of suicide substrates, isoniazid (ISD) and hydralazine (HDL), which are known inactivators of the enzyme [10,11]. We also describe a novel bimolecular rate constant that formally describes the activating effect of a suicide donor substrate on hydroperoxideinitiated inactivation of peroxidases.…”

Fluorometric Determination of Quinolinic Acid Using the Catalytic Activity of Horseradish Peroxidase

“…However, in the presence of excess H 2 O 2 relative to HRP, it is well known that other forms, such as compound III and P-670, are formed in addition to compounds I and II. [27][28][29][30] These results indicate that the concentration of H 2 O 2 in reaction solutions affect the fluorescent derivatization of QA with HRP.…”