ROLES OF EFFICIENT SUBSTRATES IN ENHANCEMENT OF PEROXIDASE-CATALYZED OXIDATIONS

Efficient peroxidase substrates may have a critical role in the oxidat ion of secondary compounds by peroxidases. Hydrazines are often oxidiz ed slowly by peroxidases due, in part, to hydrazine-dependent inactiva tion of these enzymes. Peroxidase-catalyzed oxidation of hydrazines ma y be dramatically affected by an efficient peroxidase substrate. We in vestigated this hypothesis in a model system using the well-known pero xidase substrate chlorpromazine (CPZ) and the hydrazine derivative iso niazid. CPZ stimulated isoniazid oxidation as measured by nitroblue te trazolium (NET) reduction and O-2 consumption. The kinetics of isoniaz id and CPZ oxidation by horseradish peroxidase (HRP) in the presence o f both compounds suggested CPZ was acting as an electron transfer medi ator between HRP and isoniazid. Indeed, CPZ(.+), the product of CPZ ox idation by HRP, was able to oxidize isoniazid. The rate constant for t his 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 inactivat ion of HRP was observed. The inactivation process involved the formati on of compound LII followed by accumulation of irreversibly inactivate d HRP. CPZ completely inhibited inactivation. Thus, by acting as a red ox mediator and preventing HRP inactivation, CPZ stimulated isoniazid oxidation by several orders of magnitude. Similarly, other efficient p eroxidase substrates, such as phenol and tyrosine, were also able to d ramatically stimulate isoniazid oxidation by HRP. We suggest that the presence of efficient peroxidase substrates may potentiate the activat ion of isoniazid and other hydrazines. As such, these substrates may h ave a vital role in the pharmacological and toxicological properties o f hydrazines and other compounds.