Untersuchungen zur Struktur und Funktion der Glutathionsynthetase bei der Spalthefe Schizosaccharomyces pombe
type: CreativeWork
description: In the literature the enzyme glutathione synthetase of the fission yeast S. pombe had been described - in contrast to the homodimeric enzymes of other eucaryotic organisms - as a heterotetramer composed of two „large” 33 kDa and two „small” 26 kDa subunits. The „large” subunit had been assigned to the 3' region of the GSH2 gene. The sequences of the 56 kDa protein encoded by the full length S. pombe GSH2 open reading frame and glutathione synthetases from other eukaryotes show high levels of homology over the whole alignment. Based on this alignment and on the x-ray coordinates of the human enzyme, a structural model of the fission yeast glutathione synthetase was produced. According to this model, the structure of the fission yeast protein is very similar to its human ortholog. The amino acid residues essential for binding of the substrates and cofactors are highly conserved between the two enzymes. The only striking difference involves a 15 amino acid segment (residues 204 - 218), which only exists in the fission yeast protein. The S. pombe GSH2 gene was cloned, and a histidine-tag was attached to the C-terminus of the protein. The protein was expressed in S. pombe and purified by two-step affinity chromatography. The recovered enzyme occurred in two different forms: a homodimeric protein consisting of two identical 56 kDa subunits and a heterotetrameric protein composed of two „small” 24 kDa and two „large” 32 kDa subfragments. Both variants showed glutathione synthetase activity. Both the homodimer and the heterotetramer are encoded by the GSH2 gene. The 56 kDa subunit corresponds to the complete GSH2 open reading frame. By MALDI-TOF-MS and peptide mapping, the „small” subfragment was assigned to the 5' area of the open reading frame. The 24 kDa and the 32 kDa proteins are produced following proteolytic cleavage of the 56 kDa protein. The 24 kDa protein represents the N-terminal, the 32 kDa protein the C-terminal subfragment. Protease inhibition experiments show…
abstract: In the literature the enzyme glutathione synthetase of the fission yeast S. pombe had been described - in contrast to the homodimeric enzymes of other eucaryotic organisms - as a heterotetramer composed of two „large” 33 kDa and two „small” 26 kDa subunits. The „large” subunit had been assigned to the 3' region of the GSH2 gene. The sequences of the 56 kDa protein encoded by the full length S. pombe GSH2 open reading frame and glutathione synthetases from other eukaryotes show high levels of homology over the whole alignment. Based on this alignment and on the x-ray coordinates of the human enzyme, a structural model of the fission yeast glutathione synthetase was produced. According to this model, the structure of the fission yeast protein is very similar to its human ortholog. The amino acid residues essential for binding of the substrates and cofactors are highly conserved between the two enzymes. The only striking difference involves a 15 amino acid segment (residues 204 - 218), which only exists in the fission yeast protein. The S. pombe GSH2 gene was cloned, and a histidine-tag was attached to the C-terminus of the protein. The protein was expressed in S. pombe and purified by two-step affinity chromatography. The recovered enzyme occurred in two different forms: a homodimeric protein consisting of two identical 56 kDa subunits and a heterotetrameric protein composed of two „small” 24 kDa and two „large” 32 kDa subfragments. Both variants showed glutathione synthetase activity. Both the homodimer and the heterotetramer are encoded by the GSH2 gene. The 56 kDa subunit corresponds to the complete GSH2 open reading frame. By MALDI-TOF-MS and peptide mapping, the „small” subfragment was assigned to the 5' area of the open reading frame. The 24 kDa and the 32 kDa proteins are produced following proteolytic cleavage of the 56 kDa protein. The 24 kDa protein represents the N-terminal, the 32 kDa protein the C-terminal subfragment. Protease inhibition experiments show…
author: Phlippen, Nadine
language: ger
keywords: info:eu-repo/classification/ddc/570, Biowissenschaften, Biologie, Glutathionsynthetase, Glutathion, Molecular Modelling, Proteinfaltung, Proteinevolution, zirkuläre Permutation
date published: 2003
date modified: 2026-07-30T19:42:31Z
provider: RWTH Aachen University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Publikationsserver der RWTH Aachen University