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Rutas hacia la catálisis covalente por selección reactiva para los nucleófilos de proteínas nacientes

Andrey V Reshetnyak¹, Maria Francesca Armentano, Natalia A Ponomarenko

¹Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, 16/10, Miklukho-Maklaya str, Moscow, 117871, Russia.

Journal of the American Chemical Society

|November 30, 2007

Resumen

Este estudio utilizó la selección covalente para desarrollar nuevos catalizadores enzimáticos a partir de fragmentos de anticuerpos. Los investigadores identificaron residuos específicos de tirosina que permiten el enlace covalente, lo que sugiere un nuevo enfoque para crear sitios catalíticos primitivos.

Área de la Ciencia:

La bioquímica es la bioquímica.
Biología Molecular Biología Molecular
Ingeniería de proteínas Ingeniería de proteínas.

Sus antecedentes:

La selección basada en la reactividad enriquece las bibliotecas combinatorias de biocatalizadores con funciones alteradas.
La evolución artificial de las enzimas puede proceder sin sitios catalíticos predefinidos.
Los complejos covalentes son frecuentes en los mecanismos enzimáticos, ofreciendo una base para la selección.

Objetivo del estudio:

Explorar el alcance y el mecanismo de la reactividad química en bibliotecas moleculares ingenuas utilizando selección covalente.
Para aplicar la selección covalente a través del intercambio de ésteres de fosfonato a las bibliotecas de fragmentos variables de anticuerpos (scFv).
Investigar el potencial de la selección covalente para generar nuevos biocatalizadores.

Principales métodos:

Se construyó una biblioteca de visualización de fagos de fragmentos variables de anticuerpos (scFv).
La selección covalente se realizó utilizando el intercambio de ésteres de fosfonato.
Se analizaron los clones seleccionados de scFv para las relaciones estructurales y la reactividad.
El análisis estructural identificó motivos y residuos específicos de tirosina involucrados en la unión covalente.

Principales resultados:

Los clones escFv seleccionados se segregan en enlaces covalentes y no covalentes.
Los clones de reacción covalente utilizaron exclusivamente los residuos de tirosina como nucleófilos.
Se identificaron dos motivos estructurales distintos, reclutando diferentes residuos de tirosina (Tyr32 y Tyr36).
El clon A.17, reaccionando en Tyr36, mostró una mayor cinética de fosforilación y actividad de amidasa.

Conclusiones:

La selección covalente es una estrategia viable para la evolución de la catálisis enzimática.
Los residuos de tirosina juegan un papel clave en la reactividad covalente de los scFv seleccionados.
Los motivos identificados y el clon A.17 sugieren la aparición de sitios catalíticos primitivos.
Este enfoque proporciona acceso a moléculas de proteínas que se aproximan a los primeros aparatos de catálisis covalente.

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