Structural studies have suggested that the glutathione S-transferase (GST) A1-1 isozyme contains a dynamic C-terminus which undergoes a ligand-dependent disorder-order transition and sequesters substrates within the active site. Here, the contribution of the C-terminus to the kinetics and thermodynamics of ligand binding and dissociation has been determined. Steady-state turnover rates of the wild type (WT) and a C-terminal truncated (Delta209-222) rGST A1-1 with ethacrynic acid (EA) were measured in the presence of variable concentrations of viscogen. The results indicate that a physical step involving segmental protein motion is at least partially rate limiting at temperatures between 10 and 40 degrees C for WT. Dissociation rates of the glutathione-ethacrynic acid product conjugate (GS-EA), determined by stopped-flow fluorescence, correspond to the steady-state turnover rates. In contrast, the chemical step governs the turnover reaction by Delta209-222, suggesting that the slow rate of product release for WT is controlled by the dynamics of the C-terminal coil-helix transition. In addition, the association reaction of WT rGST A1-1 with GS-EA established that the binding was biphasic and included ligand docking followed by slow isomerization of the enzyme-ligand complex. In contrast, binding of GS-EA to Delta209-222 was a monophasic, bimolecular reaction. These results indicate that the binding of GS-EA to WT rGST A1-1 proceeds via an induced fit mechanism, with a slow conformational step that corresponds to the coil-helix transition. However, the biphasic dissociation kinetics for the wild type, and the recovered kinetic parameters, suggest that a significant fraction of the [GST.GS-EA] complex ( approximately 15%) retains a persistent disordered state at equilibrium.
The polybrominated diphenyl ethers (PBDEs) are a group of flame retardants whose residues have markedly increased in the environment and in human tissues during the last decade. Of the various congeners, BDE 47 (2,2',4,4'-tetrabromodiphenyl ether) is typically the predominant congener observed in fish and wildlife samples, as well as in human tissues. Several studies indicate in utero transfer of PBDEs during pregnancy with residues accumulating in fetal tissues, and thus the potential for BDE 47-mediated injury in utero is of concern. In this study, we examined the mechanisms of BDE 47-mediated injury to primary human fetal liver hematopoietic stem cells (HSCs), which comprise a large proportion of fetal hepatic cells and play a key role in hematopoiesis during fetal development. Incubation of fetal liver HSCs with BDE 47 led to a loss of mitochondrial membrane potential and the onset of apoptosis. These effects were observed in the low micromolar range of BDE 47 exposures. At higher concentrations, BDE 47 elicited a loss of viability, which was accompanied by the generation of reactive oxygen species and peroxidation of HSC lipids. Preincubation of fetal liver HSCs with N-acetylcysteine, a glutathione (GSH) precursor, caused an increase in cellular GSH concentrations, restored mitochondrial redox status, and ameliorated the toxicity of BDE 47. BDE 47-mediated cytotoxicity or oxidative injury was not evident at the lower concentrations (< 1microM). Collectively, these data support a role for oxidative stress in the cytotoxicity of BDE 47 and indicate that oxidative stress-associated biomarkers may be useful in assessing the sublethal effects of BDE 47 toxicity in other models. However, the fact that BDE 47 undergoes a concentration-dependent accumulation in other primary cells in media that can underestimate cellular concentrations (W. R. Mundy et al., 2004, Toxicol. Sci. 82, 164-169) suggests that the HSC cell injury observed in our study may be of less relevance to human in utero PBDE exposures.
Cytochrome P450 monooxygenases (CYPs) metabolize nearly all drugs and toxins. Recently, it has become clear that CYPs exhibit both homotropic and heterotropic allosteric kinetics for many substrates. However, the mechanism of cooperative kinetics has not been established for any specific human CYP/substrate combination. Suggested mechanisms include binding of multiple substrates within distinct, static, subsites of a single large active site or binding of multiple substrates within a single fluid active site. CYP3A4 hydroxylates pyrene with positive cooperativity. Therefore, experiments were designed to exploit the fluorescence properties of pyrene, which diagnostically distinguish between pyrene.pyrene complexes versus spatially separated pyrene substrates. Pyrene complexes (excimers) yield an emission spectrum clearly distinct from pyrene monomers. In lipid-free aqueous/glycerol solutions of CYP3A4, addition of pyrene affords a concentration-dependent low-spin to high-spin conversion of the CYP3A4 heme prosthetic group, indicating occupancy of the active site by pyrene. Under the same conditions, in the presence of CYP3A4 but not other heme proteins, the excimer/monomer ratio (E/M) of pyrene was decreased in emission spectra, compared to pyrene alone. However, excitation spectra indicate a CYP3A4-dependent increase in the wavelength shift for the excimer excitation spectrum versus the monomer excitation spectrum, as well as changes in the excimer excitation peak shape and vibronic structure. These changes are reversed by the CYP3A4 substrate testosterone. Together, the results demonstrate that pyrene.pyrene ground-state complexes occupy the CYP3A4 active site, and they provide the first spectroscopic evidence for substrate complexes within a single fluid active site. Functional implications include the possibility that turnover rate, regioselectivity, and stereoselectivity of the reaction are determined by the substrate.substrate complex rather than individual substrates.
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