REGULATION OF HEME METABOLISM IN THE LIVER

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Heme is the prosthetic moiety of the hepatic hemoproteins Cytochromes P450 (P450) and tryptophan pyrrolase (TO), which are committed to metabolism/detoxification of endobiotics and xenobiotics (P450s), and oxidative tryptophan metabolism (TO). However, not all P450-mediated reactions are beneficial. Certain P450s through one electron oxidations, convert some substrates to acutely toxic radicals that escape the active site and elicit acute/chronic pathologies. Other P450s may favor non-radical pathways for the same substrates. Yet other P450s, in the course of such oxidations incur "suicide", mechanism-based inactivations, with destruction of prosthetic heme and/or apoprotein. Our studies indicate that not only the oxidative pathways favored by each P450 differ, but also that the mode of suicide inactivation by a particular substrate differs among P450s. Because the heme of all P450s is identical, they implicate the apoP450 active site structure in dictating their catalytic preferences. In this, P450IIIA1 is notorious both for its highly deviant catalytic behaviour and its overt digressions in otherwise highly conserved active site structure. Site-directed mutagenesis studies are proposed to determine the influence of these natural mutations on P450IIIA1-catalyses of radical-producing substrates, (DDEP and organic hydroperoxides), and its unusual suicide inactivation via irreversible heme binding to the apoprotein. Studies are also proposed to elucidate whether other presumed mechanism-based inactivations (P450IIIB1 by secobarbital, P450IIC11 by spironolactone), actually occur at the P450 active site and thus truly qualify as suicidal. Because after suicidal heme destruction, certain P450s (P450IIC11) incur immunochemically detectable loss, which is hemin-preventable and ATP-dependent, it is proposed that their heme-stripping and loss might promote their phosphorylation and/or ubiquitination as a prelude to their proteolytic disposal. Indeed, preliminary findings indicate that DDEP-inactivated P450IIC11 in isolated rat hepatocytes is phosphorylated. Studies are proposed to determine whether this is a feature of heme-stripped P450IIC11, to characterize its time-course, identify the phosphorylation sites and determine the heme-regulation of this process. Studies on heme-mediated regulation of rat hepatic TO will be extended: The marked functional TO loss resulting from immunochemically detectable structural loss after acute heme depletion, suggests that prosthetic heme loss destabilizes apoTO. However, not only TO loss persists in acute heme deficiency, but TO induction by glucocorticoids is also impaired. Studies are proposed to determine whether this impairment reflects instability of the newly synthesized apoTO in the absence of heme, or whether, in common with hepatic P450s, heme also regulates TO transcription/translation. It is believed, that collectively, these studies will provide insight into the structure-function relationships, the structural features that commit P450s to toxification/detoxification pathways and thus influence xenobiotic-mediated toxicity, and the ill-understood heme regulation of the turnover of these important hemoproteins.