Roles of Efficient Substrates in Enhancement of Peroxidase-Catalyzed Oxidations

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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) × 104 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.