title: Location of substrate binding sites within the integral membrane protein microsomal glutathione transferase-1.
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.
authors.affiliation: Department of Biochemistry, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
authors.forename: Laura S
authors.initials: LS
authors.lastname: Busenlehner
authors.forename: Johan
authors.initials: J
authors.lastname: Alander
authors.forename: Caroline
authors.initials: C
authors.lastname: Jegerscöhld
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Holm
authors.forename: Priyaranjan
authors.initials: P
authors.lastname: Bhakat
authors.forename: Hans
authors.initials: H
authors.lastname: Hebert
authors.forename: Ralf
authors.initials: R
authors.lastname: Morgenstern
authors.forename: Richard N
authors.initials: RN
authors.lastname: Armstrong
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D005227:Fatty Acids / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism; D006602:Hexosyltransferases / Q000737:chemistry* / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D005227:Fatty Acids; D008565:Membrane Proteins; D013438:Sulfhydryl Compounds; D006602:Hexosyltransferases; C099573:STT3A protein, human; D005982:Glutathione Transferase