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- Busenlehner, Laura S., et al.
(författare)
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Location of substrate binding sites within the integral membrane protein microsomal glutathione transferase-1
- 2007
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 46:10, s. 2812-2822
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Tidskriftsartikel (refereegranskat)abstract
- Microsomal glutathione transferase-1 (MGST1) is a trimeric, membrane-bound enzyme with both glutathione (GSH) transferase and hydroperoxidase activities. As a member of the MAPEG superfamily, MGST1 aids in the detoxication of numerous xenobiotic substrates and in cellular protection from oxidative stress through the GSH-dependent reduction of phospholipid hydroperoxides. However, little is known about the location of the different substrate binding sites, including whether the transferase and peroxidase activities overlap structurally. Although molecular density attributed to GSH has been observed in the 3.2 A resolution electron crystallographic structure of MGST1, the electrophilic and phospholipid hydroperoxide substrate binding sites remain elusive. Amide H-D exchange kinetics and H-D ligand footprinting experiments indicate that GSH and hydrophobic substrates bind within similar, but distinct, regions of MGST1. Site-directed mutagenesis, guided by the H-D exchange results, demonstrates that specific residues within the GSH footprint effect transferase activity toward 1-chloro-2,4-dinitrobenzene. In addition, cytosolic residues surrounding the chemical stress sensor C49 but not modeled in the crystal structure appear to play an important role in the formation of the binding site for hydrophobic substrates. Although the fatty acid/phospholipid binding site structurally overlaps that for GSH, it does not appear to be localized to the same region as other hydrophobic substrates. Finally, H-D exchange mass spectrometry reveals a specific conformational transition that may mediate substrate binding and/or product release. Such structural changes in MGST1 are essential for activation of the enzyme and are important for its biological function.
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- Kuang, Qie, et al.
(författare)
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Dead-end complex, lipid interactions and catalytic mechanism of microsomal glutathione transferase 1, an electron crystallography and mutagenesis investigation
- 2017
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two aliphatic chains and one glutathione (GSH) molecule. The functional unit is a homotrimer centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer supported by new in vitro mutagenesis data. Mutation of Arginine 130 to alanine resulted in complete loss of activity consistent with a role for Arginine 130 in stabilizing the strongly nucleophilic GSH thiolate required for catalysis. Based on the new model and an electron diffraction data set from crystals soaked with trinitrobenzene, that forms a dead-end Meisenheimer complex with GSH, a difference map was calculated. The map reveals side chain movements opening a cavity that defines the second substrate site.
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- Spahiu, Linda, et al.
(författare)
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Global Kinetic Mechanism of Microsomal Glutathione Transferase 1 and Insights into Dynamic Enzyme Activation
- 2017
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Ingår i: Biochemistry. - : AMER CHEMICAL SOC. - 0006-2960 .- 1520-4995. ; 56:24, s. 3089-3098
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Tidskriftsartikel (refereegranskat)abstract
- Microsomal glutathione transferase 1 (MGST1) has a unique ability to be activated, <= 30-fold, by modification with sulfhydryl reagents. MGST1 exhibits one-third-of-the-sites reactivity toward glutathione and hence heterogeneous binding to different active sites in the homotrimer. Limited turnover stopped-flow kinetic measurements of the activated enzyme allowed us to more accurately determine the KD for the "third" low-affinity GSH binding site (1.4 +/- 0.3 mM). The rate of thiolate formation, k(2) (0.77 +/- 0.06 s(-1)), relevant to turnover, could also be determined. By deriving the steadystate rate equation for a random sequential mechanism for MGST1, we can predict K-M, k(cat), and k(cat)/K-M values from these and previously determined pre-steady-state rate constants (all determined at 5 C). To assess whether the pre-steady-state behavior can account for the steady-state kinetic behavior, we have determined experimental values for kinetic parameters at 5 degrees C. For reactive substrates and the activated enzyme, data for the microscopic steps account for the global mechanism of MGST1. For the unactivated enzyme and more reactive electrophilic substrates, pre steady -state and steady-state data can be reconciled only if a more active subpopulation of MGST1 is assumed. We suggest that unactivated MGST1 can be partially activated in its unmodified form. The existence of an activated subpopulation (approximately 10%) could be demonstrated in limited turnover experiments. We therefore suggest that MSGT1 displays a preexisting dynamic equilibrium between high- and low-activity forms.
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