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[[Image:1zvj.gif|left|200px]]<br /><applet load="1zvj" size="350" color="white" frame="true" align="right" spinBox="true"
caption="1zvj, resolution 2.03&Aring;" />
'''Structure of Kumamolisin-AS mutant, D164N'''<br />


==Overview==
==Structure of Kumamolisin-AS mutant, D164N==
Kumamolisin-As is an acid collagenase with a subtilisin-like fold. Its active site contains a unique catalytic triad, Ser278-Glu78-Asp82, and a putative transition-state stabilizing residue, Asp164. In this study, the mutants D164N and E78H/D164N were engineered in order to replace parts of the catalytic machinery of kumamolisin-As with the residues found in the equivalent positions in subtilisin. Unlike the wild-type and D164N proenzymes, which undergo instantaneous processing to produce their 37-kDa mature forms, the expressed E78H/D164N proenzyme exists as an equilibrated mixture of the nicked and intact forms of the precursor. X-ray crystallographic structures of the mature forms of the two mutants showed that, in each of them, the catalytic Ser278 makes direct hydrogen bonds with the side chain of Asn164. In addition, His78 of the double mutant is distant from Ser278 and Asp82, and the catalytic triad no longer exists. Consistent with these structural alterations around the active site, these mutants showed only low catalytic activity (relative k(cat) at pH 4.0 1.3% for D164N and 0.0001% for E78H/D164N). pH-dependent kinetic studies showed that the single D164N substitution did not significantly alter the logk(cat) vs. pH and log(k(cat)/Km) vs. pH profiles of the enzyme. In contrast, the double mutation resulted in a dramatic switch of the logk(cat) vs. pH profile to one that was consistent with catalysis by means of the Ser278-His78 dyad and Asn164, which may also account for the observed ligation/cleavage equilibrium of the precursor of E78H/D164N. These results corroborate the mechanistic importance of the glutamate-mediated catalytic triad and oxyanion-stabilizing aspartic acid residue for low-pH peptidase activity of the enzyme.
<StructureSection load='1zvj' size='340' side='right'caption='[[1zvj]], [[Resolution|resolution]] 2.03&Aring;' scene=''>
 
== Structural highlights ==
==About this Structure==
<table><tr><td colspan='2'>[[1zvj]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Alicyclobacillus_sendaiensis Alicyclobacillus sendaiensis]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1ZVJ OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1ZVJ FirstGlance]. <br>
1ZVJ is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Alicyclobacillus_sendaiensis Alicyclobacillus sendaiensis] with <scene name='pdbligand=CA:'>CA</scene> and <scene name='pdbligand=SO4:'>SO4</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1ZVJ OCA].  
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.03&#8491;</td></tr>
 
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CA:CALCIUM+ION'>CA</scene>, <scene name='pdbligand=SO4:SULFATE+ION'>SO4</scene></td></tr>
==Reference==
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=1zvj FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1zvj OCA], [https://pdbe.org/1zvj PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1zvj RCSB], [https://www.ebi.ac.uk/pdbsum/1zvj PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1zvj ProSAT]</span></td></tr>
Processing, catalytic activity and crystal structures of kumamolisin-As with an engineered active site., Okubo A, Li M, Ashida M, Oyama H, Gustchina A, Oda K, Dunn BM, Wlodawer A, Nakayama T, FEBS J. 2006 Jun;273(11):2563-76. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=16704427 16704427]
</table>
== Function ==
[https://www.uniprot.org/uniprot/Q8GB88_9BACL Q8GB88_9BACL]
== 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/zv/1zvj_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=1zvj ConSurf].
<div style="clear:both"></div>
__TOC__
</StructureSection>
[[Category: Alicyclobacillus sendaiensis]]
[[Category: Alicyclobacillus sendaiensis]]
[[Category: Single protein]]
[[Category: Large Structures]]
[[Category: Gustchina, A.]]
[[Category: Gustchina A]]
[[Category: Li, M.]]
[[Category: Li M]]
[[Category: Nakayama, T.]]
[[Category: Nakayama T]]
[[Category: Wlodawer, A.]]
[[Category: Wlodawer A]]
[[Category: CA]]
[[Category: SO4]]
[[Category: d164n]]
[[Category: kumamolisin-as]]
 
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 16:19:28 2008''

Latest revision as of 09:06, 14 February 2024

Structure of Kumamolisin-AS mutant, D164N

1zvj, resolution 2.03Å

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