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Christopher R Pudney¹, Richard S K Lane, Alistair J Fielding

¹Manchester Institute of Biotechnology and Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.

Journal of the American Chemical Society

|February 14, 2013

Resumen

Los efectos de isótopos cinéticos de una sola molécula (KIEs) utilizando spFRET revelan detalles de la reacción enzimática. Este método separa la transferencia enzimática de H de la dinámica de las proteínas y los fluoróforos, lo que hace avanzar la comprensión de la catálisis.

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Área de la Ciencia:

La bioquímica es la bioquímica.
La cinética de las enzimas.
Química física es la química física de las cosas.

Sus antecedentes:

Las mediciones conjuntas de los efectos de isótopos cinéticos (KIEs) han avanzado la comprensión de las reacciones catalizadas por enzimas, pero se enfrentan a limitaciones.
Los KIEs son cruciales para estudiar los pasos limitadores de velocidad, el túnel cuántico, la dinámica y los estados reactivos múltiples en las enzimas.

Objetivo del estudio:

Explorar las KIE enzimáticas de molécula única (SM) para obtener nuevos conocimientos sobre la catálisis enzimática.
Aplicar la transferencia de energía de fluorescencia de un solo par (spFRET) para medir las PYMES KIE en reacciones de transferencia de H.

Principales métodos:

Se utilizó la transferencia de energía de fluorescencia de un solo par (spFRET) para medir las PYMES.
Métodos desarrollados y evaluados para la extracción de PYMES KIEs de spFRET trazas de tiempo.
Se ha investigado la transferencia de H catalizada por el pentaeritritol tetranitrato reductasa.

Principales resultados:

Se midieron con éxito los KIEs enzimáticos de SM para las reacciones de transferencia de H.
Demostró la capacidad del análisis SM KIE para diferenciar entre procesos enzimáticos y no enzimáticos.
Contribuciones separadas de la dinámica de las proteínas, el comportamiento de los fluoróforos y la propia reacción de transferencia de H.

Conclusiones:

El análisis KIE de una sola molécula proporciona un nuevo y poderoso enfoque para estudiar la catálisis enzimática.
Este método permite la desconvolución de la química de reacción de la dinámica intrínseca de las proteínas.
Ofrece una vía para resolver controversias de larga data en la cinética de las enzimas.