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==Aspartate Aminotransferase Active Site Mutant V39F/N194A== | |||
<StructureSection load='1ix8' size='340' side='right'caption='[[1ix8]], [[Resolution|resolution]] 2.20Å' scene=''> | |||
| | == Structural highlights == | ||
<table><tr><td colspan='2'>[[1ix8]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1IX8 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1IX8 FirstGlance]. <br> | |||
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.2Å</td></tr> | |||
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=PLP:PYRIDOXAL-5-PHOSPHATE'>PLP</scene></td></tr> | |||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=1ix8 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1ix8 OCA], [https://pdbe.org/1ix8 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1ix8 RCSB], [https://www.ebi.ac.uk/pdbsum/1ix8 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1ix8 ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[https://www.uniprot.org/uniprot/AAT_ECOLI AAT_ECOLI] | |||
== Evolutionary Conservation == | |||
[[Image:Consurf_key_small.gif|200px|right]] | |||
Check<jmol> | |||
<jmolCheckbox> | |||
<scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/ix/1ix8_consurf.spt"</scriptWhenChecked> | |||
<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked> | |||
<text>to colour the structure by Evolutionary Conservation</text> | |||
</jmolCheckbox> | |||
</jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=1ix8 ConSurf]. | |||
<div style="clear:both"></div> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Aspartate aminotransferase has been known to undergo a significant conformational change, in which the small domain approaches the large domain, and the residues at the entrance of the active site pack together, on binding of substrates. Accompanying this conformational change is a two-unit increase in the pK(a) of the pyridoxal 5'-phosphate-Lys(258) aldimine, which has been proposed to enhance catalysis. To elucidate how the conformational change is coupled to the shift in the aldimine pK(a) and how these changes are involved in catalysis, we analyzed structurally and kinetically an enzyme in which Val(39) located at both the domain interface and the entrance of the active site was replaced with a bulkier residue, Phe. The V39F mutant enzyme showed a more open conformation, and the aldimine pK(a) was lowered by 0.7 unit compared with the wild-type enzyme. When Asn(194) had been replaced by Ala in advance, the V39F mutation did not decrease the aldimine pK(a), showing that the domain rotation controls the aldimine pK(a) via the Arg(386)-Asn(194)-pyridoxal 5'-phosphate linkage system. The maleate-bound V39F enzyme showed the aldimine pK(a) 0.9 unit lower than that of the maleate-bound wild-type enzyme. However, the positions of maleate, Asn(194), and Arg(386) were superimposable between the mutant and the wild-type enzymes; therefore, the domain rotation was not the cause of the lowered aldimine pK(a) value. The maleate-bound V39F enzyme showed an altered side-chain packing pattern in the 37-39 region, and the lack of repulsion between Gly(38) carbonyl O and Tyr(225) Oeta seemed to be the cause of the reduced pK(a) value. Kinetic analysis suggested that the repulsion increases the free energy level of the Michaelis complex and promotes the catalytic reaction. | |||
Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis.,Hayashi H, Mizuguchi H, Miyahara I, Nakajima Y, Hirotsu K, Kagamiyama H J Biol Chem. 2003 Mar 14;278(11):9481-8. Epub 2002 Dec 17. PMID:12488449<ref>PMID:12488449</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 1ix8" style="background-color:#fffaf0;"></div> | |||
== | ==See Also== | ||
Aspartate aminotransferase | *[[Aspartate aminotransferase 3D structures|Aspartate aminotransferase 3D structures]] | ||
== References == | |||
== | <references/> | ||
__TOC__ | |||
</StructureSection> | |||
[[Category: Escherichia coli]] | [[Category: Escherichia coli]] | ||
[[Category: | [[Category: Large Structures]] | ||
[[Category: Hayashi | [[Category: Hayashi H]] | ||
[[Category: Hirotsu | [[Category: Hirotsu K]] | ||
[[Category: Kagamiyama | [[Category: Kagamiyama H]] | ||
[[Category: Miyahara | [[Category: Miyahara I]] | ||
[[Category: Mizuguchi | [[Category: Mizuguchi H]] | ||
[[Category: Nakajima | [[Category: Nakajima Y]] | ||
Latest revision as of 02:38, 28 December 2023
Aspartate Aminotransferase Active Site Mutant V39F/N194AAspartate Aminotransferase Active Site Mutant V39F/N194A
Structural highlights
FunctionEvolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedAspartate aminotransferase has been known to undergo a significant conformational change, in which the small domain approaches the large domain, and the residues at the entrance of the active site pack together, on binding of substrates. Accompanying this conformational change is a two-unit increase in the pK(a) of the pyridoxal 5'-phosphate-Lys(258) aldimine, which has been proposed to enhance catalysis. To elucidate how the conformational change is coupled to the shift in the aldimine pK(a) and how these changes are involved in catalysis, we analyzed structurally and kinetically an enzyme in which Val(39) located at both the domain interface and the entrance of the active site was replaced with a bulkier residue, Phe. The V39F mutant enzyme showed a more open conformation, and the aldimine pK(a) was lowered by 0.7 unit compared with the wild-type enzyme. When Asn(194) had been replaced by Ala in advance, the V39F mutation did not decrease the aldimine pK(a), showing that the domain rotation controls the aldimine pK(a) via the Arg(386)-Asn(194)-pyridoxal 5'-phosphate linkage system. The maleate-bound V39F enzyme showed the aldimine pK(a) 0.9 unit lower than that of the maleate-bound wild-type enzyme. However, the positions of maleate, Asn(194), and Arg(386) were superimposable between the mutant and the wild-type enzymes; therefore, the domain rotation was not the cause of the lowered aldimine pK(a) value. The maleate-bound V39F enzyme showed an altered side-chain packing pattern in the 37-39 region, and the lack of repulsion between Gly(38) carbonyl O and Tyr(225) Oeta seemed to be the cause of the reduced pK(a) value. Kinetic analysis suggested that the repulsion increases the free energy level of the Michaelis complex and promotes the catalytic reaction. Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis.,Hayashi H, Mizuguchi H, Miyahara I, Nakajima Y, Hirotsu K, Kagamiyama H J Biol Chem. 2003 Mar 14;278(11):9481-8. Epub 2002 Dec 17. PMID:12488449[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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