| Registro completo |
| Provedor de dados: |
BJMBR
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| País: |
Brazil
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| Título: |
Characterization of the interdependency between residues that bind the substrate in a β-glycosidase
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| Autores: |
Tomassi,M.H.
Rozenfeld,J.H.K.
Gonçalves,L.M.
Marana,S.R.
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| Data: |
2010-01-01
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| Ano: |
2010
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| Palavras-chave: |
Β-glycosidase
Substrate specificity
Site-directed mutagenesis
Spodoptera frugiperda
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| Resumo: |
The manner by which effects of simultaneous mutations combine to change enzymatic activity is not easily predictable because these effects are not always additive in a linear manner. Hence, the characterization of the effects of simultaneous mutations of amino acid residues that bind the substrate can make a significant contribution to the understanding of the substrate specificity of enzymes. In the β-glycosidase from Spodoptera frugiperda (Sfβgly), both residues Q39 and E451 interact with the substrate and this is essential for defining substrate specificity. Double mutants of Sfβgly (A451E39, S451E39 and S451N39) were prepared by site-directed mutagenesis, expressed in bacteria and purified using affinity chromatography. These enzymes were characterized using p-nitrophenyl β-galactoside and p-nitrophenyl β-fucoside as substrates. The k cat/Km ratio for single and double mutants of Sfβgly containing site-directed mutations at positions Q39 and E451 was used to demonstrate that the effect on the free energy of ES‡ (enzyme-transition state complex) of the double mutations (∆∆G‡xy) is not the sum of the effects resulting from the single mutations (∆∆G‡x and ∆∆G‡y). This difference in ∆∆G‡ indicates that the effects of the single mutations partially overlap. Hence, this common effect counts only once in ∆∆G‡xy. Crystallographic data on β-glycosidases reveal the presence of a bidentate hydrogen bond involving residues Q39 and E451 and the same hydroxyl group of the substrate. Therefore, both thermodynamic and crystallographic data suggest that residues Q39 and E451 exert a mutual influence on their respective interactions with the substrate.
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| Tipo: |
Info:eu-repo/semantics/other
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| Idioma: |
Inglês
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| Identificador: |
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2010000100002
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| Editor: |
Associação Brasileira de Divulgação Científica
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| Relação: |
10.1590/S0100-879X2009007500033
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| Formato: |
text/html
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| Fonte: |
Brazilian Journal of Medical and Biological Research v.43 n.1 2010
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| Direitos: |
info:eu-repo/semantics/openAccess
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