Bioactivation of lamotrigine in vivo in rat and in vitro in human liver microsomes, hepatocytes, and epidermal keratinocytes: characterization of thioether conjugates by liquid chromatography/mass spectrometry and high field nuclear magnetic resonance spectroscopy.

Previous studies suggested that lamotrigene (LTG) underwent bioactivation to a reactive aryl epoxide intermediate in rats. Nevertheless, definitive structures of these thioether conjugates, which are often needed to substantiate the mechanism of bioactivation and identity of reactive intermediate(s), were not fully established. In the present study, GSH, cysteinylglycine, and N-acetyl cysteine conjugates of LTG were isolated from bile of rats orally dosed with LTG (100 mg/kg), and their structures were fully elucidated by LC/MS and NMR. The definitive structural characterization of these metabolites provided evidence for the existence of a reactive aryl epoxide that was trapped as a GSH adduct. In vitro studies using various hepatic cellular and subcellular fractions obtained from human and rat were performed to demonstrate that LTG underwent bioactivation to form a GSH conjugate that was identical to the one initially characterized from in vivo studies. Human P450 2A6 and rat P450 2C11 appeared to be the primary enzymes activating LTG in human and rat liver microsomes, respectively. Interindividual variation in the bioactivation of LTG was demonstrated with 20 individual human liver microsomes. Furthermore, it was shown that human epidermal keratinocytes were capable of forming the same GSH conjugate, suggesting that LTG could be bioactivated in skin cells. The results from these studies suggest that LTG has the potential to undergo hepatic and nonhepatic bioactivation, leading to a reactive aryl epoxide intermediate in human. The bioactivation of LTG in epidermal cells provides a possible explanation for the idiosyncratic cutaneous reactions associated with LTG therapy.

The regulatory role of residues 226-232 in phosphoenolpyruvate carboxylase from maize.

The regulatory properties of maize phosphoenolpyruvate carboxylase were significantly altered by site-directed mutagenesis of residues 226 through 232. This conserved sequence element, RTDEIRR, is part of a surface loop at the dimer interface. Mutation of individual residues in this sequence caused various kinetic changes, including desensitization of the enzyme to key allosteric effectors or alteration of the K(0.5 PEP) for the substrate phosphoenolpyruvate. R231A, and especially R232Q, displayed decreased apparent affinity for the activator glucose-6-phosphate. Apparent affinity for the activator glycine was reduced in D228N and R232Q, while the maximum activation caused by glycine was greatly reduced in R226Q and E229A. R226Q and E229A also showed significantly lower sensitivity to the inhibitors malate and aspartate. E229A exhibited a low K(0.5 PEP), while the K(0.5 PEP )of R232Q was significantly higher than that of wild type. Thus these seven residues are critical determinants of the enzyme's kinetic responses to activators, inhibitors and substrate. The present results support an earlier suggestion that Arg 231 contributes to the binding site of the allosteric activator glucose-6-phosphate, and are consistent with other proposals that the substrate phosphoenolpyruvate allosterically activates the enzyme by binding at or near the glucose-6-phosphate site. The results also suggest that the glycine binding site may be contiguous with the glucose-6-phosphate binding site. Glu 229, which extends from this interface region through the interior of the protein and emerges near the aspartate binding site, may provide a physical link for propagating conformational changes between the allosteric activator and inhibitor binding regions.