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Mecanismo y residuos del sitio activo de GDP-fucosa sintasa
Stephen T B Lau1, Martin E Tanner
1Department of Chemistry, University of British Columbia, Vancouver, British Columbia, V6T 1Z1 Canada.
Journal of the American Chemical Society
|December 5, 2008
Resumen
Este estudio revela el mecanismo paso a paso de la GDP-fucosa sintasa (GFS) en la producción de GDP-L-fucosa. Los residuos clave Cys109 y His179 se identifican como catalizadores ácido/base cruciales para las etapas de epimerización.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Enzimología Enzimología.
- Glucobiología Glucobiología.
Sus antecedentes:
- La L-fucosa es vital para los glucoconjugados como los antígenos de los grupos sanguíneos.
- La biosíntesis de GDP-L-fucosa es esencial para los procesos celulares.
- La GDP-fucosa sintasa (GFS) cataliza el paso final en la producción de GDP-L-fucosa.
Objetivo del estudio:
- Elucidar el orden de epimerización en las reacciones GDP-fucosa sintasa (GFS).
- Identificar las funciones de los residuos del sitio activo en la catalización de epimerizaciones.
- Comprender el mecanismo de conversión de la GDP-4-ceto-6-desoxi-D-manosa a la GDP-L-fucosa.
Principales métodos:
- Mutagénesis dirigida al sitio de la GDP-fucosa sintasa (GFS).
- Estudios del efecto del isótopo cinético utilizando sustratos etiquetados con deuterio.
- Análisis de los productos de reacción y la formación de productos intermedios.
Principales resultados:
- El mutante Cys109Ser produce GDP-6-deoxy-D-altrosa, lo que indica que la epimerización C-3' precede a la epimerización C-5'.
- Cys109 actúa como base para la desprotonación C-3', confirmada por los efectos de isótopos cinéticos.
- El mutante His179Gln facilita el lavado de deuterio, implicando a His179 como un catalizador ácido.
Conclusiones:
- La secuencia de epimerización está ordenada: C-3' seguido de C-5''.
- Cys109 funciona como una base, y His179 funciona como un ácido en ambas etapas de epimerización.
- Esta visión mecanicista avanza en nuestra comprensión del metabolismo de la L-fucosa y la biosíntesis del glucoconjugado.
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