2gtu: Difference between revisions
New page: left|200px<br /> <applet load="2gtu" size="450" color="white" frame="true" align="right" spinBox="true" caption="2gtu, resolution 2.55Å" /> '''LIGAND-FREE HUMAN G... |
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[[Image:2gtu.gif|left|200px]]<br /> | [[Image:2gtu.gif|left|200px]]<br /><applet load="2gtu" size="350" color="white" frame="true" align="right" spinBox="true" | ||
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caption="2gtu, resolution 2.55Å" /> | caption="2gtu, resolution 2.55Å" /> | ||
'''LIGAND-FREE HUMAN GLUTATHIONE S-TRANSFERASE M2-2 (E.C.2.5.1.18), MONOCLINIC CRYSTAL FORM'''<br /> | '''LIGAND-FREE HUMAN GLUTATHIONE S-TRANSFERASE M2-2 (E.C.2.5.1.18), MONOCLINIC CRYSTAL FORM'''<br /> | ||
==Overview== | ==Overview== | ||
A series of chimeric human Mu class glutathione S-transferases were | A series of chimeric human Mu class glutathione S-transferases were designed to determine mechanisms by which they activate enzyme-bound glutathione (GSH) for reaction with electrophilic substrates. In view of evidence that the His(107) residue of hGSTM1a-1a is important for catalysis (Patskovsky, Y. V., Patskovska, L. N., and Listowsky, I. (1999) Biochemistry 38, 1193-1202), the cognate Arg(107) residue of the hGSTM2 subunit was replaced (R107N or R107H) and arginine residues were also incorporated into position 107 of hGSTM1 (H107R) and hGSTM4 (S107R) subunits. The major distinguishing kinetic properties invariably associated with enzymes containing an Arg(107) residue include an inverse dependence of k(cat) on viscosity and lower K(m(GSH values relative to enzymes with other residues at that position. Moreover, affinities for GSH thiolate anion binding are greater for enzymes containing Arg(107))), with K(d) values of 20-50 microM that are consistent with the K(m(GSH values (10-25 microM) obtained by steady-state kinetic analyses. Both thermodynamic and kinetic and data indicate that the Arg(107))) residue is specifically involved in enhancing the binding affinity of GSH thiolate anion relative to that of the protonated form. These enzymes therefore, can be more effective at lower GSH concentrations. Combined mutations indicate that both Arg(107) and Tyr(6) residues are required for thiolate anion formation and stabilization. The three-dimensional structure of ligand-free hGSTM2-2 determined by x-ray crystallography suggests that Arg(107) maintains an electrostatic interaction with the Asp(161) side chain (3 A apart), but is distant from the GSH-binding site. However, an alternative energetically favorable model places the guanidino group 4 A from the sulfur atom of bound GSH. It is suggested therefore, that in solution, motion of the positively charged arginine into the catalytic pocket could provide a counter ion to promote ionization of the sulfhydryl group of GSH, thereby accounting for the observed greater affinity of enzymes containing Arg(107) for binding of thiolate anion. | ||
==About this Structure== | ==About this Structure== | ||
2GTU is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens]. Active as [http://en.wikipedia.org/wiki/Glutathione_transferase Glutathione transferase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.5.1.18 2.5.1.18] Full crystallographic information is available from [http:// | 2GTU is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens]. Active as [http://en.wikipedia.org/wiki/Glutathione_transferase Glutathione transferase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.5.1.18 2.5.1.18] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2GTU OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: Homo sapiens]] | [[Category: Homo sapiens]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Almo, S | [[Category: Almo, S C.]] | ||
[[Category: Fedorov, A | [[Category: Fedorov, A A.]] | ||
[[Category: Listowsky, I.]] | [[Category: Listowsky, I.]] | ||
[[Category: Patskovska, L | [[Category: Patskovska, L N.]] | ||
[[Category: Patskovsky, Y | [[Category: Patskovsky, Y V.]] | ||
[[Category: conjugation]] | [[Category: conjugation]] | ||
[[Category: cytosolic]] | [[Category: cytosolic]] | ||
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[[Category: transferase]] | [[Category: transferase]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:35:21 2008'' | ||
Revision as of 18:35, 21 February 2008
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LIGAND-FREE HUMAN GLUTATHIONE S-TRANSFERASE M2-2 (E.C.2.5.1.18), MONOCLINIC CRYSTAL FORM
OverviewOverview
A series of chimeric human Mu class glutathione S-transferases were designed to determine mechanisms by which they activate enzyme-bound glutathione (GSH) for reaction with electrophilic substrates. In view of evidence that the His(107) residue of hGSTM1a-1a is important for catalysis (Patskovsky, Y. V., Patskovska, L. N., and Listowsky, I. (1999) Biochemistry 38, 1193-1202), the cognate Arg(107) residue of the hGSTM2 subunit was replaced (R107N or R107H) and arginine residues were also incorporated into position 107 of hGSTM1 (H107R) and hGSTM4 (S107R) subunits. The major distinguishing kinetic properties invariably associated with enzymes containing an Arg(107) residue include an inverse dependence of k(cat) on viscosity and lower K(m(GSH values relative to enzymes with other residues at that position. Moreover, affinities for GSH thiolate anion binding are greater for enzymes containing Arg(107))), with K(d) values of 20-50 microM that are consistent with the K(m(GSH values (10-25 microM) obtained by steady-state kinetic analyses. Both thermodynamic and kinetic and data indicate that the Arg(107))) residue is specifically involved in enhancing the binding affinity of GSH thiolate anion relative to that of the protonated form. These enzymes therefore, can be more effective at lower GSH concentrations. Combined mutations indicate that both Arg(107) and Tyr(6) residues are required for thiolate anion formation and stabilization. The three-dimensional structure of ligand-free hGSTM2-2 determined by x-ray crystallography suggests that Arg(107) maintains an electrostatic interaction with the Asp(161) side chain (3 A apart), but is distant from the GSH-binding site. However, an alternative energetically favorable model places the guanidino group 4 A from the sulfur atom of bound GSH. It is suggested therefore, that in solution, motion of the positively charged arginine into the catalytic pocket could provide a counter ion to promote ionization of the sulfhydryl group of GSH, thereby accounting for the observed greater affinity of enzymes containing Arg(107) for binding of thiolate anion.
About this StructureAbout this Structure
2GTU is a Single protein structure of sequence from Homo sapiens. Active as Glutathione transferase, with EC number 2.5.1.18 Full crystallographic information is available from OCA.
ReferenceReference
The enhanced affinity for thiolate anion and activation of enzyme-bound glutathione is governed by an arginine residue of human Mu class glutathione S-transferases., Patskovsky YV, Patskovska LN, Listowsky I, J Biol Chem. 2000 Feb 4;275(5):3296-304. PMID:10652317
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