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Cinética de la abstracción del átomo de hidrógeno del sustrato por un radical tiilo del sitio activo en la

Lisa Olshansky¹, Arturo A Pizano, Yifeng Wei

¹Department of Chemistry and Chemical Biology, Harvard University , 12 Oxford Street, Cambridge, Massachusetts 02138, United States.

Journal of the American Chemical Society

|October 30, 2014

Resumen

Este estudio revela el paso limitador de velocidad en la catálisis de la ribonucleotida reductasa (RNR) mediante el uso de un enfoque fotoquímico. La propagación radical iniciada por la luz desenmascaró la escisión del enlace 3'-C-H como el paso más lento en la transferencia de electrones acoplados a protones (PCET).

Área de la Ciencia:

Bioquímica y enzimología.
Química de los radicales de las proteínas.
Bioenergética y transferencia de electrones.

Sus antecedentes:

Las reductasas de ribonucleótidos (RNR) son enzimas esenciales que catalizan la conversión de nucleótido a desoxinucleótido.
Las RNR de Clase Ia utilizan una vía compleja de transferencia de electrones acoplados a protones (PCET) que involucra aminoácidos redox-activos durante ~35 Å.
Los cambios de conformación pueden oscurecer la cinética intrínseca del PCET en RNRs.

Objetivo del estudio:

Para investigar el paso limitador de la velocidad en la vía PCET de E. coli clase Ia RNR.
Para desacoplar la cinética PCET de los cambios conformacionales más lentos utilizando un enfoque fotoquímico.
Observar y cuantificar espectroscópicamente los radicales intermedios clave y los eventos de transferencia.

Principales métodos:

Incorporación específica del sitio de un [Re(I) ] fotooxidante en un RNR modificado ([Re]-β2).
Sustitución de la tirosina Y356 por 2,3,5-trifluorotirosina para permitir la generación de radicales.
Espectroscopia de absorción transitoria para monitorear la cinética de desintegración de radicales tirosílicos bajo diversas condiciones (sustrato, variantes de proteínas, etiquetado isotópico).

Principales resultados:

La generación fotoquímica de radicales tirosilo permitió la observación de los eventos PCET.
El aumento de la descomposición radical, indicativo de PCET avanzada, se observó solo con RNR de tipo silvestre y sustrato sin etiqueta.
La hendidura del enlace 3'-C-H del sustrato por el radical tiilo C439 se identificó como el paso que limita la velocidad en el PCET avanzado.

Conclusiones:

El paso de escisión del enlace 3'-C-H tiene un límite inferior constante de velocidad de (1.4 ± 0.4) × 10^4 s(-1).
La iniciación fotoquímica proporciona una herramienta poderosa para estudiar dinámicas radicales rápidas, de otro modo inaccesibles, en las enzimas.
Este estudio aclara la química crítica del sitio activo que rige la catálisis de RNR.

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