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alfa-1,4-Glucan liasa realiza una trans-eliminación a través de un desplazamiento nucleófilo seguido de una
Seung Seo Lee1, Shukun Yu, Stephen G Withers
1Department of Chemistry, Protein Engineering Network of Centres of Excellence of Canada, University of British Columbia, Vancouver, BC Canada V6T 1Z1.
Journal of the American Chemical Society
|May 2, 2002
Resumen
La alfa-glucano liasa (GLasa) utiliza un mecanismo de sin-eliminación, confirmado mediante la captura de un intermediario de la enzima glicosil. Este intermedio incluye Asp 553 y libera 1,5-d-anhidrofructosa.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Enzimología Enzimología.
- Química de los carbohidratos Química de los carbohidratos
Sus antecedentes:
- La alfa-glucano liasa (GLasa) cataliza la escisión de los enlaces alfa-1,4-glucosídicos.
- El mecanismo de reacción de la GLasa ha sido objeto de investigación.
Objetivo del estudio:
- Para dilucidar el mecanismo catalítico de la alfa-glucano liasa (GLasa).
- Para identificar el residuo de aminoácidos clave involucrado en el proceso catalítico.
- Para diferenciar entre los mecanismos de eliminación propuestos.
Principales métodos:
- La inactivación de la GLasa mediante el uso de 5-fluoro-beta-l-idosil fluoruro, un inactivador de la alfa-glucosidasa.
- Aislamiento y caracterización del intermediario atrapado de la glicosila-enzima.
- Identificación del residuo de aminoácido nucleófilo a través del análisis de secuencia.
- Estudios del efecto del isótopo cinético utilizando sustratos sustituidos por deuterio.
Principales resultados:
- La GLasa fue inactivada, y un intermediario estable de la glicosil-enzima fue atrapado con éxito.
- El ácido aspártico en la posición 553 (Asp 553) fue identificado como el residuo nucleófilo, conservado en las alfa-glucosidasas relacionadas.
- El intermediario se somete a una reacción de sin-eliminación para obtener el producto 1,5-d-anhidrofructosa.
- La ausencia de un efecto de isótopo cinético primario en C2 indicado contra un mecanismo de trans-eliminación.
Conclusiones:
- El mecanismo catalítico de la GLasa implica una vía de eliminación sin.
- Asp 553 es crucial para la catálisis, formando un intermediario covalente.
- Los hallazgos aclaran la vía de reacción de la GLasa y su relación con otras alfa-glucosidasas.
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