The mechanism of the amidases: mutating the glutamate adjacent to the catalytic triad inactivates the enzyme due to substrate mispositioning

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dc.contributor.authorWeber, Brandon W
dc.contributor.authorKimani, Serah W
dc.contributor.authorVarsani, Arvind
dc.contributor.authorCowan, Donald A
dc.contributor.authorHunter, Roger
dc.contributor.authorVenter, Gerhard A
dc.contributor.authorGumbart, James C
dc.contributor.authorSewell, Trevor B
dc.date.accessioned2016-09-02T10:44:19Z
dc.date.available2016-09-02T10:44:19Z
dc.date.issued2013
dc.date.updated2016-09-02T10:43:03Z
dc.description.abstractAll known nitrilase superfamily amidase and carbamoylase structures have an additional glutamate thatis hydrogen bonded to the catalytic lysine in addition to the Glu, Lys, Cys “catalytic triad.” In the amidase from Geobacillus pallidus, mutating this glutamate (Glu-142) to a leucine or aspartate renders the enzyme inactive. X-ray crystal structure determination shows that the structural integrity of the enzymeismaintained despite themutation with the catalytic cysteine (Cys-166), lysine (Lys-134), and glutamate (Glu- 59)in positions similar to those of the wild-type enzyme. In the case of the E142L mutant, a chloride ion is located in the position occupied by Glu-142 O 1 in the wild-type enzyme andinteracts with the active site lysine. In the case of the E142D mutant, this site is occupied by Asp-142 O1.In neither case is an atom located at the position of Glu-142 O 2 in the wild-type enzyme. The active site cysteine of the E142Lmutant was found to form aMichael adduct with acrylamide, which is a substrate of the wild-type enzyme, due to an interaction that places the double bond of the acrylamide rather than the amide carbonyl carbon adjacent to the active site cysteine. Our results demonstrate that in the wild-type active site a crucial role is played by the hydrogen bond between Glu-142 O 2 and the substrate amino groupin positioning the substrate with the correct stereoelectronic alignment to enable the nucleophilic attack on the carbonyl carbon by the catalytic cysteine.en_ZA
dc.identifierhttp://dx.doi.org/10.1074/jbc.M113.503284
dc.identifier.apacitationWeber, B. W., Kimani, S. W., Varsani, A., Cowan, D. A., Hunter, R., Venter, G. A., ... Sewell, T. B. (2013). The mechanism of the amidases: mutating the glutamate adjacent to the catalytic triad inactivates the enzyme due to substrate mispositioning. <i>Journal of Biological Chemistry</i>, http://hdl.handle.net/11427/21657en_ZA
dc.identifier.chicagocitationWeber, Brandon W, Serah W Kimani, Arvind Varsani, Donald A Cowan, Roger Hunter, Gerhard A Venter, James C Gumbart, and Trevor B Sewell "The mechanism of the amidases: mutating the glutamate adjacent to the catalytic triad inactivates the enzyme due to substrate mispositioning." <i>Journal of Biological Chemistry</i> (2013) http://hdl.handle.net/11427/21657en_ZA
dc.identifier.citationWeber, B. W., Kimani, S. W., Varsani, A., Cowan, D. A., Hunter, R., Venter, G. A., ... & Sewell, B. T. (2013). The Mechanism of the Amidases MUTATING THE GLUTAMATE ADJACENT TO THE CATALYTIC TRIAD INACTIVATES THE ENZYME DUE TO SUBSTRATE MISPOSITIONING. Journal of Biological Chemistry, 288(40), 28514-28523.en_ZA
dc.identifier.issn0021-9258en_ZA
dc.identifier.ris TY - Journal Article AU - Weber, Brandon W AU - Kimani, Serah W AU - Varsani, Arvind AU - Cowan, Donald A AU - Hunter, Roger AU - Venter, Gerhard A AU - Gumbart, James C AU - Sewell, Trevor B AB - All known nitrilase superfamily amidase and carbamoylase structures have an additional glutamate thatis hydrogen bonded to the catalytic lysine in addition to the Glu, Lys, Cys “catalytic triad.” In the amidase from Geobacillus pallidus, mutating this glutamate (Glu-142) to a leucine or aspartate renders the enzyme inactive. X-ray crystal structure determination shows that the structural integrity of the enzymeismaintained despite themutation with the catalytic cysteine (Cys-166), lysine (Lys-134), and glutamate (Glu- 59)in positions similar to those of the wild-type enzyme. In the case of the E142L mutant, a chloride ion is located in the position occupied by Glu-142 O 1 in the wild-type enzyme andinteracts with the active site lysine. In the case of the E142D mutant, this site is occupied by Asp-142 O1.In neither case is an atom located at the position of Glu-142 O 2 in the wild-type enzyme. The active site cysteine of the E142Lmutant was found to form aMichael adduct with acrylamide, which is a substrate of the wild-type enzyme, due to an interaction that places the double bond of the acrylamide rather than the amide carbonyl carbon adjacent to the active site cysteine. Our results demonstrate that in the wild-type active site a crucial role is played by the hydrogen bond between Glu-142 O 2 and the substrate amino groupin positioning the substrate with the correct stereoelectronic alignment to enable the nucleophilic attack on the carbonyl carbon by the catalytic cysteine. DA - 2013 DB - OpenUCT DP - University of Cape Town J1 - Journal of Biological Chemistry LK - https://open.uct.ac.za PB - University of Cape Town PY - 2013 SM - 0021-9258 T1 - The mechanism of the amidases: mutating the glutamate adjacent to the catalytic triad inactivates the enzyme due to substrate mispositioning TI - The mechanism of the amidases: mutating the glutamate adjacent to the catalytic triad inactivates the enzyme due to substrate mispositioning UR - http://hdl.handle.net/11427/21657 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/21657
dc.identifier.urihttp://www.jbc.org/content/288/40/28514.short
dc.identifier.vancouvercitationWeber BW, Kimani SW, Varsani A, Cowan DA, Hunter R, Venter GA, et al. The mechanism of the amidases: mutating the glutamate adjacent to the catalytic triad inactivates the enzyme due to substrate mispositioning. Journal of Biological Chemistry. 2013; http://hdl.handle.net/11427/21657.en_ZA
dc.languageengen_ZA
dc.publisherAmerican Society for Biochemistry and Molecular Biologyen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.sourceJournal of Biological Chemistryen_ZA
dc.source.urihttp://www.asbmb.org/page.aspx?id=598
dc.subject.otheramidases
dc.subject.othermutation
dc.subject.othercatalytic triad
dc.titleThe mechanism of the amidases: mutating the glutamate adjacent to the catalytic triad inactivates the enzyme due to substrate mispositioningen_ZA
dc.typeJournal Articleen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceArticleen_ZA
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