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Pruebas cinéticas de estado estacionario para una actividad de apertura de anillos en la fructosa-1,6-(bis) fosfato
1Biology Department, Boston University, Boston, Massachusetts 02215, USA.
Journal of the American Chemical Society
|March 18, 2004
Resumen
La cinética de la enzima fructosa 1,6-bisfosfato aldolasa (aldolasa) revela que cataliza la apertura del anillo de hexosa. Esto sugiere un posible segundo sitio activo, desafiando la comprensión previa de la vía glicolítica.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Enzimología Enzimología.
- Las vías metabólicas.
Sus antecedentes:
- La fructosa 1,6-bisfosfato aldolasa es una enzima glicolítica clave.
- Las aldolasas de clase I requieren sustratos de hexosa en una forma de cadena abierta para la formación intermedia de Schiff-base.
- La forma de cadena abierta representa una pequeña fracción del azúcar en equilibrio, y su unión a la aldoasa no se entiende completamente.
Objetivo del estudio:
- Para determinar la forma química de la fructosa 1,6-bisfosfato que se une a la aldolasa.
- Para dilucidar las etapas iniciales del ciclo catalítico de la aldolasa, incluyendo la unión al sustrato y la formación de Schiff-base.
- Investigar el papel de la aldolasa en la catalización de la apertura del anillo de su sustrato de hexosa.
Principales métodos:
- Cinética del estado transitorio utilizando experimentos de recambio único con exceso de aldolasa.
- Medición de las velocidades de reacción desde la unión inicial hasta la formación de la base de Schiff.
- Experimentos de aproximación al equilibrio para analizar la unión al sustrato y la cinética de reacción.
Principales resultados:
- La velocidad de formación de la base de Schiff excedió la velocidad de apertura del anillo no catalizado de la alfa- o beta-furanosa a 4°C.
- La aldolasa exhibió distintas tasas de unión y reacción para diferentes anomalías de la fructosa 1,6-bisfosfato.
- Una fase de reacción rápida (70% de sustrato) correlacionada con el beta-anomero, y una fase lenta (30%) con el alfa-anomero.
Conclusiones:
- La aldolasa cataliza activamente la apertura del anillo de la fructosa 1,6-bisfosfato.
- La interacción de la enzima con diferentes anomalías sugiere un reconocimiento específico del sustrato y la catálisis.
- La evidencia apunta a un posible segundo sitio activo no reconocido previamente en la aldolasa para catalizar la apertura del anillo.
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