title: Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase.
abstract: Both soluble and membrane-bound enzymes can catalyze the conversion of lipophilic substrates. The precise substrate access path, with regard to phase, has however, until now relied on conjecture from enzyme structural data only (certainly giving credible and valuable hypotheses). Alternative methods have been missing. To obtain the first experimental evidence directly determining the access paths (of lipophilic substrates) to phase constrained enzymes we here describe the application of a BODIPY-derived substrate (PS1). Using this tool, which is not accessible to cytosolic enzymes in the presence of detergent and, by contrast, not accessible to membrane embedded enzymes in the absence of detergent, we demonstrate that cytosolic and microsomal glutathione transferases (GSTs), both catalyzing the activation of PS1, do so only within their respective phases. This approach can serve as a guideline to experimentally validate substrate access paths, a fundamental property of phase restricted enzymes. Examples of other enzyme classes with members in both phases are xenobiotic-metabolizing sulphotransferases/UDP-glucuronosyl transferases or epoxide hydrolases. Since specific GSTs have been suggested to contribute to tumor drug resistance, PS1 can also be utilized as a tool to discriminate between phase constrained members of these enzymes by analyzing samples in the absence and presence of Triton X-100.
authors.affiliation: Institute of Environmental Medicine, Karolinska Institutet, Nobels väg 13, 17177, Stockholm, Sweden.
authors.forename: Marcus
authors.initials: M
authors.lastname: Cebula
authors.affiliation: UNAM-Institute of Material Science and Nanotechnology, Bilkent University, Bilkent, Ankara, 06800, Turkey.
authors.forename: Ilke Simsek
authors.initials: IS
authors.lastname: Turan
authors.affiliation: Department of Neurochemistry, Stockholm University, Svante Arrhenius väg 16C, 10691, Stockholm, Sweden.
authors.forename: Birgitta
authors.initials: B
authors.lastname: Sjödin
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, 17177, Stockholm, Sweden.
authors.forename: Madhuranayaki
authors.initials: M
authors.lastname: Thulasingam
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, 17177, Stockholm, Sweden.
authors.forename: Joseph
authors.initials: J
authors.lastname: Brock
authors.affiliation: Experimental Biomolecular Physics, Royal Institute of Technology-KTH, Albanova, Roslagsvägen 30 B, 11419, Stockholm, Sweden.
authors.forename: Volodymyr
authors.initials: V
authors.lastname: Chmyrov
authors.affiliation: Experimental Biomolecular Physics, Royal Institute of Technology-KTH, Albanova, Roslagsvägen 30 B, 11419, Stockholm, Sweden.
authors.forename: Jerker
authors.initials: J
authors.lastname: Widengren
authors.affiliation: Department of Chemistry, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Abe
authors.affiliation: Department of Neurochemistry, Stockholm University, Svante Arrhenius väg 16C, 10691, Stockholm, Sweden.
authors.forename: Bengt
authors.initials: B
authors.lastname: Mannervik
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, 17177, Stockholm, Sweden.
authors.forename: Jesper Z
authors.initials: JZ
authors.lastname: Haeggström
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, 17177, Stockholm, Sweden.
authors.forename: Agnes
authors.initials: A
authors.lastname: Rinaldo-Matthis
authors.affiliation: Department of Chemistry &UNAM-Institute of Material Science and Nanotechnology, Bilkent University, Bilkent, Ankara, 06800, Turkey.
authors.forename: Engin U
authors.initials: EU
authors.lastname: Akkaya
authors.affiliation: Institute of Environmental Medicine, Karolinska Institutet, Nobels väg 13, 17177, Stockholm, Sweden.
authors.forename: Ralf
authors.initials: R
authors.lastname: Morgenstern
mesh_terms: D000818:Animals; D055162:Biocatalysis; D001896:Boron Compounds / Q000737:chemistry; D003600:Cytosol / Q000201:enzymology; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D004851:Epoxide Hydrolases / Q000737:chemistry* / Q000378:metabolism; D005057:Eukaryotic Cells / Q000201:enzymology; D005456:Fluorescent Dyes / Q000737:chemistry; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008658:Inactivation, Metabolic; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008958:Models, Molecular; D017830:Octoxynol / Q000737:chemistry; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000737:chemistry* / Q000378:metabolism; D015262:Xenobiotics / Q000737:chemistry / Q000378:metabolism
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D004800:Enzymes, Immobilized; D005456:Fluorescent Dyes; D015262:Xenobiotics; D017830:Octoxynol; D014453:Glucuronosyltransferase; D005982:Glutathione Transferase; D015238:Sulfotransferases; D004851:Epoxide Hydrolases