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| [[Image:1ejs.gif|left|200px]]<br /><applet load="1ejs" size="350" color="white" frame="true" align="right" spinBox="true"
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| caption="1ejs, resolution 2.0Å" />
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| '''CRYSTAL STRUCTURE OF THE H219N VARIANT OF KLEBSIELLA AEROGENES UREASE'''<br />
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| ==Overview== | | ==Crystal Structure of the H219N Variant of Klebsiella Aerogenes Urease== |
| Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze urea hydrolysis at >10(14)-fold the spontaneous rate. To better define the enzyme mechanism, we examined the kinetics and structures for a suite of site-directed variants involving four residues at the active site: His320, His219, Asp221, and Arg336. Compared to wild-type urease, the H320A, H320N, and H320Q variants exhibit similar approximately 10(-)(5)-fold deficiencies in rates, modest K(m) changes, and disorders in the peptide flap covering their active sites. The pH profiles for these mutant enzymes are anomalous with optima near 6 and shoulders that extend to pH 9. H219A urease exhibits 10(3)-fold increased K(m) over that of native enzyme, whereas the increase is less marked ( approximately 10(2)-fold) in the H219N and H219Q variants that retain hydrogen bonding capability. Structures for these variants show clearly resolved active site water molecules covered by well-ordered peptide flaps. Whereas the D221N variant is only moderately affected compared to wild-type enzyme, D221A urease possesses low activity ( approximately 10(-)(3) that of native enzyme), a small increase in K(m), and a pH 5 optimum. The crystal structure for D221A urease is reminiscent of the His320 variants. The R336Q enzyme has a approximately 10(-)(4)-fold decreased catalytic rate with near-normal pH dependence and an unaffected K(m). Phenylglyoxal inactivates the R336Q variant at over half the rate observed for native enzyme, demonstrating that modification of non-active-site arginines can eliminate activity, perhaps by affecting the peptide flap. Our data favor a mechanism in which His219 helps to polarize the substrate carbonyl group, a metal-bound terminal hydroxide or bridging oxo-dianion attacks urea to form a tetrahedral intermediate, and protonation occurs via the general acid His320 with Asp221 and Arg336 orienting and influencing the acidity of this residue. Furthermore, we conclude that the simple bell-shaped pH dependence of k(cat) and k(cat)/K(m) for the native enzyme masks a more complex underlying pH dependence involving at least four pK(a)s. | | <StructureSection load='1ejs' size='340' side='right'caption='[[1ejs]], [[Resolution|resolution]] 2.00Å' scene=''> |
| | == Structural highlights == |
| | <table><tr><td colspan='2'>[[1ejs]] is a 3 chain structure with sequence from [https://en.wikipedia.org/wiki/Klebsiella_aerogenes Klebsiella aerogenes]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1EJS OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1EJS 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]] 2Å</td></tr> |
| | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=KCX:LYSINE+NZ-CARBOXYLIC+ACID'>KCX</scene>, <scene name='pdbligand=NI:NICKEL+(II)+ION'>NI</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=1ejs FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1ejs OCA], [https://pdbe.org/1ejs PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1ejs RCSB], [https://www.ebi.ac.uk/pdbsum/1ejs PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1ejs ProSAT]</span></td></tr> |
| | </table> |
| | == Function == |
| | [https://www.uniprot.org/uniprot/URE1_KLEAE URE1_KLEAE] |
| | == 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/ej/1ejs_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=1ejs ConSurf]. |
| | <div style="clear:both"></div> |
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| ==About this Structure== | | ==See Also== |
| 1EJS is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Klebsiella_aerogenes Klebsiella aerogenes] with <scene name='pdbligand=NI:'>NI</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Urease Urease], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.1.5 3.5.1.5] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1EJS OCA].
| | *[[Urease 3D structures|Urease 3D structures]] |
| | | __TOC__ |
| ==Reference==
| | </StructureSection> |
| Kinetic and structural characterization of urease active site variants., Pearson MA, Park IS, Schaller RA, Michel LO, Karplus PA, Hausinger RP, Biochemistry. 2000 Jul 25;39(29):8575-84. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=10913264 10913264]
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| [[Category: Klebsiella aerogenes]] | | [[Category: Klebsiella aerogenes]] |
| [[Category: Protein complex]] | | [[Category: Large Structures]] |
| [[Category: Urease]]
| | [[Category: Hausinger RP]] |
| [[Category: Hausinger, R P.]] | | [[Category: Karplus PA]] |
| [[Category: Karplus, P A.]] | | [[Category: Michel LO]] |
| [[Category: Michel, L O.]] | | [[Category: Park IS]] |
| [[Category: Park, I S.]] | | [[Category: Pearson MA]] |
| [[Category: Pearson, M A.]] | | [[Category: Schaller RA]] |
| [[Category: Schaller, R A.]] | |
| [[Category: NI]]
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| [[Category: alpha-beta barrel]]
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| [[Category: nickel metalloenzyme]]
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| ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:28:30 2008''
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