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Estabilización selectiva del estado de transición de la chorismato mutasa por un donante de enlace de hidrógeno
Alexander Kienhöfer1, Peter Kast, Donald Hilvert
1Laboratorium für Organische Chemie, Swiss Federal Institute of Technology, ETH Hönggerberg, CH-8093 Zürich, Switzerland.
Journal of the American Chemical Society
|March 13, 2003
Resumen
La sustitución de la arginina por la citrulina en el Bacillus subtilis chorismate mutase redujo significativamente la actividad de la enzima. Esto pone de relieve el papel esencial de la estabilización electrostática del estado de transición para una catálisis eficiente.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- La cinética de las enzimas.
- La ingeniería de proteínas es la ingeniería de proteínas.
Sus antecedentes:
- La chorismato mutasa AroH de Bacillus subtilis cataliza una reacción crucial en la vía de biosíntesis de los aminoácidos aromáticos.
- El residuo del sitio activo Arginina 90 (Arg90) es crítico para la catálisis, formando enlaces de hidrógeno con el sustrato chorismato.
Objetivo del estudio:
- Para investigar el papel de las interacciones electrostáticas en el mecanismo catalítico de AroH chorismate mutase.
- Comprender la contribución de la carga positiva de Arg90 a la estabilización del estado de transición.
Principales métodos:
- Se utilizó semisíntesis química para reemplazar el Arg90 de tipo silvestre por citrulina (Cit), creando la variante Arg90Cit.
- La cinética de las enzimas se analizó midiendo la constante de velocidad catalítica (kcat) y la constante de Michaelis (Km) tanto para las enzimas de tipo salvaje como para las variantes.
- Las interacciones enzima-inhibidor se evaluaron utilizando un inhibidor conformacionalmente restringido.
Principales resultados:
- La variante Arg90Cit mostró una disminución de más de 104 veces en kcat y un aumento de 2,7 veces en Km, lo que indica una eficiencia catalítica significativamente reducida.
- La afinidad de la variante por un inhibidor que imita el estado de transición se redujo solo unas 6 veces, lo que sugiere que el inhibidor no replica completamente la distribución de carga del estado de transición.
- Estos hallazgos demuestran que la simple complementariedad con la conformación reactiva del sustrato es insuficiente para la catálisis.
Conclusiones:
- La estabilización electrostática del estado de transición polarizado por un donante de enlace catiónico de hidrógeno es esencial para la alta eficiencia catalítica de la chorismato mutasa AroH.
- La carga positiva en Arg90 juega un papel vital en la estabilización de la carga negativa en desarrollo en el átomo de oxígeno durante la escisión del enlace C-O en el estado de transición.
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