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Análisis del estado de transición de la enolpiruvilshikimato 3-fosfato (EPSP) sintetasa (AroA) catalizada por EPSP en
Meiyan Lou1, Steven K Burger, Meghann E Gilpin
1Department of Chemistry & Chemical Biology, and †Department of Biochemistry & Biomedical Sciences, McMaster University , 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada.
Journal of the American Chemical Society
|July 7, 2012
Resumen
La catálisis enzimática de la transferencia de protones al carbono es difícil. Enolpyruvylshikimate 3-phosphate synthase (AroA) utiliza un estado de transición temprano para estabilizar un intermediario clave, superando este desafío.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- La catálisis enzimática es una catálisis enzimática.
- La cinética de las sustancias químicas.
Sus antecedentes:
- La transferencia de protones a los átomos de carbono presenta importantes obstáculos catalíticos debido a las barreras de alta energía y la termodinámica desfavorable.
- La enolpiruvilshikimato 3-fosfato sintasa (EPSP sintasa, AroA) se enfrenta al desafío de protonar el carbono metileno del fosfoenolpiruvato (EPSP) en su reacción inversa.
Objetivo del estudio:
- Para analizar el estado de transición de la hidrólisis de EPSP catalizada por AroA, que sirve como un análogo para la reacción inversa de la enzima.
- Para investigar el mecanismo de la protonación de carbono de metileno e identificar las estrategias catalíticas de AroA.
Principales métodos:
- Utilizó efectos de isótopos cinéticos (KIEs), incluidos los KIEs de 3-(14) C y de deuterio disolvente, para el análisis del estado de transición.
- Desarrolló un sistema de eliminación de fosfato inorgánico para administrar las concentraciones ubicuas de fosfato.
- Empleado el modelado computacional para analizar las estructuras de estado de transición y comparar con datos experimentales.
Principales resultados:
- La reacción procede paso a paso a través de la protonación C3 irreversible, formando un intermediario catiónico EPSP (mecanismo AH(‡) *AN).
- Un gran experimento 3-(14) C KIE (1.032 ± 0.005) indicó un fuerte acoplamiento entre C3 y el movimiento de protones.
- Se observó un KIE de deuterio disolvente muy bajo (0.97 ± 0.04), consistente con un estado de transición temprano.
- Un gran KIE de 2-(14) C reveló la estabilización de la carga positiva C2 por un "sándwich electrostático" de Asp313 y Glu341.
Conclusiones:
- AroA facilita la catálisis cambiando el estado de transición antes que las reacciones catalizadas por ácido, logrando un cambio Hammond significativo.
- La estrategia catalítica de la enzima consiste en estabilizar la carga positiva del intermediario catiónico EPSP.
- Los modelos computacionales corroboran los hallazgos experimentales, destacando el papel de los residuos del sitio activo en la catálisis.
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