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[[Image: | ==Structure of S102T E. coli Alkaline Phosphatase-phosphate intermediate at 2.20A resolution== | ||
<StructureSection load='2ga3' size='340' side='right' caption='[[2ga3]], [[Resolution|resolution]] 2.20Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[2ga3]] is a 2 chain structure with sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2GA3 OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=2GA3 FirstGlance]. <br> | |||
</td></tr><tr><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=MG:MAGNESIUM+ION'>MG</scene>, <scene name='pdbligand=SO4:SULFATE+ION'>SO4</scene>, <scene name='pdbligand=ZN:ZINC+ION'>ZN</scene><br> | |||
<tr><td class="sblockLbl"><b>[[Non-Standard_Residue|NonStd Res:]]</b></td><td class="sblockDat"><scene name='pdbligand=TPO:PHOSPHOTHREONINE'>TPO</scene></td></tr> | |||
<tr><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[2g9y|2g9y]]</td></tr> | |||
<tr><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">phoA ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=562 Escherichia coli])</td></tr> | |||
<tr><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/Alkaline_phosphatase Alkaline phosphatase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.3.1 3.1.3.1] </span></td></tr> | |||
<tr><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=2ga3 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=2ga3 OCA], [http://www.rcsb.org/pdb/explore.do?structureId=2ga3 RCSB], [http://www.ebi.ac.uk/pdbsum/2ga3 PDBsum]</span></td></tr> | |||
<table> | |||
== 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/ga/2ga3_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/chain_selection.php?pdb_ID=2ata ConSurf]. | |||
<div style="clear:both"></div> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
We report here the construction of a mutant version of Escherichia coli alkaline phosphatase (AP) in which the active site Ser was replaced by Thr (S102T), in order to investigate whether the enzyme can utilize Thr as the nucleophile and whether the rates of the critical steps in the mechanism are altered by the substitution. The mutant AP with Thr at position 102 exhibited an approximately 4000-fold decrease in k(cat) along with a small decrease in Km. The decrease in catalytic efficiency of approximately 2000-fold was a much smaller drop than that observed when Ala or Gly were substituted at position 102. The mechanism by which Thr can substitute for Ser in AP was further investigated by determining the X-ray structure of the S102T enzyme in the presence of the Pi (S102T_Pi), and after soaking the crystals with substrate (S102T_sub). In the S102T_Pi structure, the Pi was coordinated differently with its position shifted by 1.3 A compared to the structure of the wild-type enzyme in the presence of Pi. In the S102T_sub structure, a covalent Thr-Pi intermediate was observed, instead of the expected bound substrate. The stereochemistry of the phosphorus in the S102T_sub structure was inverted compared to the stereochemistry in the wild-type structure, as would be expected after the first step of a double in-line displacement mechanism. We conclude that the S102T mutation resulted in a shift in the rate-determining step in the mechanism allowing us to trap the covalent intermediate of the reaction in the crystal. | |||
Trapping the tetrahedral intermediate in the alkaline phosphatase reaction by substitution of the active site serine with threonine.,Wang J, Kantrowitz ER Protein Sci. 2006 Oct;15(10):2395-401. PMID:17008720<ref>PMID:17008720</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
==See Also== | ==See Also== | ||
*[[Alkaline phosphatase|Alkaline phosphatase]] | *[[Alkaline phosphatase|Alkaline phosphatase]] | ||
== References == | |||
== | <references/> | ||
< | __TOC__ | ||
</StructureSection> | |||
[[Category: Alkaline phosphatase]] | [[Category: Alkaline phosphatase]] | ||
[[Category: Escherichia coli]] | [[Category: Escherichia coli]] | ||
Revision as of 06:52, 29 September 2014
Structure of S102T E. coli Alkaline Phosphatase-phosphate intermediate at 2.20A resolutionStructure of S102T E. coli Alkaline Phosphatase-phosphate intermediate at 2.20A resolution
Structural highlights
Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedWe report here the construction of a mutant version of Escherichia coli alkaline phosphatase (AP) in which the active site Ser was replaced by Thr (S102T), in order to investigate whether the enzyme can utilize Thr as the nucleophile and whether the rates of the critical steps in the mechanism are altered by the substitution. The mutant AP with Thr at position 102 exhibited an approximately 4000-fold decrease in k(cat) along with a small decrease in Km. The decrease in catalytic efficiency of approximately 2000-fold was a much smaller drop than that observed when Ala or Gly were substituted at position 102. The mechanism by which Thr can substitute for Ser in AP was further investigated by determining the X-ray structure of the S102T enzyme in the presence of the Pi (S102T_Pi), and after soaking the crystals with substrate (S102T_sub). In the S102T_Pi structure, the Pi was coordinated differently with its position shifted by 1.3 A compared to the structure of the wild-type enzyme in the presence of Pi. In the S102T_sub structure, a covalent Thr-Pi intermediate was observed, instead of the expected bound substrate. The stereochemistry of the phosphorus in the S102T_sub structure was inverted compared to the stereochemistry in the wild-type structure, as would be expected after the first step of a double in-line displacement mechanism. We conclude that the S102T mutation resulted in a shift in the rate-determining step in the mechanism allowing us to trap the covalent intermediate of the reaction in the crystal. Trapping the tetrahedral intermediate in the alkaline phosphatase reaction by substitution of the active site serine with threonine.,Wang J, Kantrowitz ER Protein Sci. 2006 Oct;15(10):2395-401. PMID:17008720[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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