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La cooperatividad inducida por una sola mutación en la interfaz de subunidad de una enzima dimérica: la glutatión
N S Scrutton1, M P Deonarain, A Berry
1Department of Biochemistry, University of Cambridge, United Kingdom.
Science (New York, N.Y.)
|November 13, 1992
Resumen
Una sola mutación en Escherichia coli glutatión reductasa puede inducir la cooperatividad en la función de la enzima. Interrumpir la interfaz del dímero con una mutación crea un interruptor molecular, afectando la cinética de la enzima y la unión del sustrato.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- La cinética de las enzimas.
- La estructura de las proteínas es la estructura de las proteínas.
Sus antecedentes:
- La glutatión reductasa de Escherichia coli es una enzima homodimérica crucial para el equilibrio redox celular.
- Comprender los mecanismos que controlan la cooperación de las enzimas es vital para la ingeniería enzimática y el desarrollo de fármacos.
Objetivo del estudio:
- Para investigar el papel de la interfaz dímero en la regulación del comportamiento alostérico de la E. coli glutatión reductasa.
- Para determinar si una mutación en la interfaz del dímero puede conferir cooperatividad a la enzima.
Principales métodos:
- La mutagénesis dirigida al sitio se utilizó para reemplazar la glicina 418 con triptófano en la E. coli glutatión reductasa.
- Se realizaron ensayos de cinética enzimática para medir la afinidad y la cooperatividad de unión al glutatión (coeficiente de Hill).
- Los experimentos de heterodimerización se llevaron a cabo mezclando subunidades de tipo salvaje y mutantes.
Principales resultados:
- La mutación de glicina418 a triptófano dio como resultado una enzima altamente cooperativa (coeficiente de Hill 1.76) para la unión al glutatión.
- La cooperatividad fue abolida en los heterodímeros formados por una subunidad mutante y una subunidad de tipo salvaje.
- La mutación interrumpió el empaque atómico en la interfaz del dímero, alterando el mecanismo cinético de la enzima.
Conclusiones:
- Una sola mutación distante del sitio activo puede actuar como un interruptor molecular para inducir la cooperación enzimática.
- La interfaz del dímero juega un papel crítico en la regulación alosterica de la E. coli glutatión reductasa.
- La interrupción de las interacciones proteína-proteína puede alterar significativamente la función y la cinética de las enzimas.
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