title: Naturally occurring Phe151Leu substitution near a conserved folding module lowers stability of glutathione transferase P1-1.
abstract: Glutathione transferases (GSTs) are a family of enzymes that detoxify electrophilic compounds, such as carcinogens or drugs, by conjugating them to glutathione. The enzymes have contributed to the understanding of protein structure, due to large differences in amino acid sequence within the family, yet similar architecture and folding. Our objective was to conduct a systematic survey of GSTP1 polymorphisms and their function. Nearly all variants detected were known polymorphisms: IVS4+13C>A; Ile105Val; Ala114Val; and g.2596T>C (Ser185Ser). However, we also found a novel Phe151Leu substitution in an African-American subject (1 out of 111). Kinetic parameters for the conjugation reaction with 1-chloro-2,4-dinitrobenzene (CDNB) were determined for the novel variant enzyme purified via heterologous expression in Escherichia coli. Five substrates were used for measurement of specific activities, including isothiocyanate compounds that occur in cruciferous vegetables (benzylisothiocyanate, phenethylisothiocyanate, and sulforaphane). Such isothiocyanate substrates are potential cancer chemopreventive agents that are conjugated by GSTs. No major change in kinetic parameters was observed. However, the half-life at 50 degrees C of the Leu 151 enzyme was reduced to 12 min, as compared to 28 min for the Phe 151 enzyme. Residue 151 is located at the N-terminus of helix alpha6 in GST motif II, surrounded by hydrophobic residues, and near the conserved "hydrophobic staple" and N-capping box motifs. These local structural elements aid in formation of helix alpha6 and promote proper folding and protein stability. Analysis of the three-dimensional structure showed that substitution of Phe 151 with Leu produces a hydrophobic cavity in the GSTP1 core, thereby destabilizing its structure. Phe151Leu represents one of the first-described allelic variations in a protein folding motif.
authors.affiliation: Department of Pediatrics and the Research and Education Institute, Harbor-UCLA Medical Center, 1124 W Carson St, Torrance, CA 90502, USA. henry_lin@humc.edu
authors.forename: Henry J
authors.initials: HJ
authors.lastname: Lin
authors.forename: Ann Sofie
authors.initials: AS
authors.lastname: Johansson
authors.forename: Gun
authors.initials: G
authors.lastname: Stenberg
authors.forename: Alicia M
authors.initials: AM
authors.lastname: Materi
authors.forename: Jae Man
authors.initials: JM
authors.lastname: Park
authors.forename: Aihua
authors.initials: A
authors.lastname: Dai
authors.forename: Haiyan
authors.initials: H
authors.lastname: Zhou
authors.forename: Jason S Y
authors.initials: JS
authors.lastname: Gim
authors.forename: Irving H
authors.initials: IH
authors.lastname: Kau
authors.forename: Steven I
authors.initials: SI
authors.lastname: Hardy
authors.forename: Michael W
authors.initials: MW
authors.lastname: Parker
authors.forename: Bengt
authors.initials: B
authors.lastname: Mannervik
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D044383:Black People / Q000235:genetics; D004137:Dinitrochlorobenzene / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D014644:Genetic Variation; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000235:genetics; D008958:Models, Molecular; D010649:Phenylalanine / Q000235:genetics; D011110:Polymorphism, Genetic; D017510:Protein Folding; D001741:Black or African American
chemical_list: D004137:Dinitrochlorobenzene; D007527:Isoenzymes; D010649:Phenylalanine; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase; D007930:Leucine