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CRYSTAL STRUCTURE OF ISOCITRATE DEHYDROGENASE MUTANT K230M BOUND TO ISOCITRATE AND MN2+

OverviewOverview

Isocitrate dehydrogenase catalyses the two step, acid base, oxidative decarboxylation of isocitrate to alpha-ketoglutarate. Lysine 230 was suggested to act as proton donor based on geometry and spatial proximity to isocitrate. To clarify further the role of lysine 230, we co-crystallized the lysine-to-methionine mutant (K230M) with isocitrate and with alpha-ketoglutarate. Crystals were flash-frozen and the two structures were determined and refined to 2. 1 A. Several new features were identified relative to the wild-type structure. Seven side-chains previously unplaced in the wild-type structure were identified and included in the model, and the amino acid terminus was extended by an alanine residue. Many additional water molecules were identified.Examination of the K230M active sites (K230M isocitrate and K230M-ketoglutarate) revealed that tyrosine 160 protrudes further into the active site in the presence of either isocitrate or alpha-ketoglutarate in K230 M than it does in the wild-type structure. Also, methionine 230 was not as fully extended, and asparagine 232 rotates approximately 30 degrees toward the ligand permitting polar interactions. Outside the active site cleft a tetragonal volume of density was identified as a sulfate molecule. Its location and interactions suggest it may influence the equilibrium between the tetragonal and the orthorhombic forms of isocitrate dehydrogenase. Differences observed in the active site water structure between the wild-type and K230M structures were due to a single point mutation. A water molecule was located in the position equivalent to that occupied by the wild-type epsilon-amine of lysine 230; a water molecule in that location in K230M suggests it may influence catalysis in the mutant. Comparison of K230M complexed with isocitrate and alpha-ketoglutarate illuminates the influence a ligand has on active site water structure.

About this StructureAbout this Structure

1CW1 is a Single protein structure of sequence from Escherichia coli with , and as ligands. Active as Isocitrate dehydrogenase (NADP(+)), with EC number 1.1.1.42 Full crystallographic information is available from OCA.

ReferenceReference

Active site water molecules revealed in the 2.1 A resolution structure of a site-directed mutant of isocitrate dehydrogenase., Cherbavaz DB, Lee ME, Stroud RM, Koshland DE Jr, J Mol Biol. 2000 Jan 21;295(3):377-85. PMID:10623532

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 '''CRYSTAL STRUCTURE OF ISOCITRATE DEHYDROGENASE MUTANT K230M BOUND TO ISOCITRATE AND MN2+'''<br />
 ==Overview==
-Isocitrate dehydrogenase catalyses the two step, acid base, oxidative, decarboxylation of isocitrate to alpha-ketoglutarate. Lysine 230 was, suggested to act as proton donor based on geometry and spatial proximity, to isocitrate. To clarify further the role of lysine 230, we, co-crystallized the lysine-to-methionine mutant (K230M) with isocitrate, and with alpha-ketoglutarate. Crystals were flash-frozen and the two, structures were determined and refined to 2. 1 A. Several new features, were identified relative to the wild-type structure. Seven side-chains, previously unplaced in the wild-type structure were identified and, included in the model, and the amino acid terminus was extended by an, alanine residue. Many additional water molecules were, identified.Examination of the K230M active sites (K230M isocitrate and, K230M-ketoglutarate) revealed that tyrosine 160 protrudes further into the, active site in the presence of either isocitrate or alpha-ketoglutarate in, K230 M than it does in the wild-type structure. Also, methionine 230 was, not as fully extended, and asparagine 232 rotates approximately 30 degrees, toward the ligand permitting polar interactions. Outside the active site, cleft a tetragonal volume of density was identified as a sulfate molecule., Its location and interactions suggest it may influence the equilibrium, between the tetragonal and the orthorhombic forms of isocitrate, dehydrogenase. Differences observed in the active site water structure, between the wild-type and K230M structures were due to a single point, mutation. A water molecule was located in the position equivalent to that, occupied by the wild-type epsilon-amine of lysine 230; a water molecule in, that location in K230M suggests it may influence catalysis in the mutant., Comparison of K230M complexed with isocitrate and alpha-ketoglutarate, illuminates the influence a ligand has on active site water structure.
+Isocitrate dehydrogenase catalyses the two step, acid base, oxidative decarboxylation of isocitrate to alpha-ketoglutarate. Lysine 230 was suggested to act as proton donor based on geometry and spatial proximity to isocitrate. To clarify further the role of lysine 230, we co-crystallized the lysine-to-methionine mutant (K230M) with isocitrate and with alpha-ketoglutarate. Crystals were flash-frozen and the two structures were determined and refined to 2. 1 A. Several new features were identified relative to the wild-type structure. Seven side-chains previously unplaced in the wild-type structure were identified and included in the model, and the amino acid terminus was extended by an alanine residue. Many additional water molecules were identified.Examination of the K230M active sites (K230M isocitrate and K230M-ketoglutarate) revealed that tyrosine 160 protrudes further into the active site in the presence of either isocitrate or alpha-ketoglutarate in K230 M than it does in the wild-type structure. Also, methionine 230 was not as fully extended, and asparagine 232 rotates approximately 30 degrees toward the ligand permitting polar interactions. Outside the active site cleft a tetragonal volume of density was identified as a sulfate molecule. Its location and interactions suggest it may influence the equilibrium between the tetragonal and the orthorhombic forms of isocitrate dehydrogenase. Differences observed in the active site water structure between the wild-type and K230M structures were due to a single point mutation. A water molecule was located in the position equivalent to that occupied by the wild-type epsilon-amine of lysine 230; a water molecule in that location in K230M suggests it may influence catalysis in the mutant. Comparison of K230M complexed with isocitrate and alpha-ketoglutarate illuminates the influence a ligand has on active site water structure.
 ==About this Structure==
-CW1 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with MN, SO4 and ICT as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Isocitrate_dehydrogenase_(NADP(+)) Isocitrate dehydrogenase (NADP(+))], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.42 1.1.1.42] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1CW1 OCA].
+CW1 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=MN:'>MN</scene>, <scene name='pdbligand=SO4:'>SO4</scene> and <scene name='pdbligand=ICT:'>ICT</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Isocitrate_dehydrogenase_(NADP(+)) Isocitrate dehydrogenase (NADP(+))], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.42 1.1.1.42] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1CW1 OCA].
 ==Reference==
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 [[Category: oxidoreductase]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 12:47:49 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:10:19 2008''