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Impacto de la mutación en las reacciones de transferencia de protones en la isomerasa cetosteroide: ideas de
Dhruva K Chakravorty1, Sharon Hammes-Schiffer
1Department of Chemistry, 104 Chemistry Building, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Journal of the American Chemical Society
|May 11, 2010
Resumen
Las mutaciones en los residuos del sitio activo de la cetosteroide isomerasa (KSI) Tyr14 y Asp99 reducen las tasas catalíticas al desestabilizar el intermediario dienolato. Las simulaciones revelan un enlace de hidrógeno alterado e interacciones electrostáticas en los mutantes, lo que afecta la eficiencia de la transferencia de protones.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Enzimología Enzimología.
- Química computacional es la química computacional.
Sus antecedentes:
- La isomerasa cetosteroide (KSI) cataliza las reacciones esenciales de transferencia de protones.
- Los enlaces de hidrógeno de Tyr14 y Asp99 estabilizan el intermediario dienolato en KSI. tipo silvestre (WT).
- Comprender las funciones de los residuos del sitio activo es crucial para el esclarecimiento del mecanismo enzimático.
Objetivo del estudio:
- Para investigar el impacto de las mutaciones Tyr14 y Asp99 en el mecanismo catalítico de KSI.
- Para analizar los cambios en el enlace de hidrógeno, conformación e interacciones durante la transferencia de protones.
- Para correlacionar los resultados de la simulación con los datos cinéticos experimentales.
Principales métodos:
- Simulaciones de dinámica molecular (DM) utilizando un modelo de enlace de valencia empírico (EVB).
- Análisis de patrones de enlaces de hidrógeno, dinámica conformacional y energías de interacción.
- Cálculo de perfiles de energía libre y constantes de velocidad para las etapas de transferencia de protones.
Principales resultados:
- Las mutaciones Y14F y D99L aumentaron significativamente la energía libre del intermediario dienolato, reduciendo la primera tasa de transferencia de protones.
- WT KSI mantuvo dos enlaces de hidrógeno en el sitio activo, mientras que los mutantes típicamente tenían uno, afectando la estabilización intermedia.
- La estabilización electrostática del intermedio se redujo en los mutantes en comparación con WT KSI.
Conclusiones:
- Las mutaciones alteran la estructura del sitio activo preorganizado de KSI, lo que lleva a una reducción de la eficiencia catalítica.
- El enlace de hidrógeno alterado y las interacciones electrostáticas en los mutantes desestabilizan el intermediario dienolato.
- Los sitios activos de la enzima requieren dinámicas conformacionales específicas para una catálisis eficiente, incluso cuando están preorganizados.
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