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Mecanismo catalítico único de la fosfolipasa C específica para el fosfatidilinositol de Streptomyces antibioticus El
Chuan Bai1, Li Zhao, Ming-Daw Tsai
1Department of Medicinal Chemistry and Pharmacognosy, The University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Journal of the American Chemical Society
|January 8, 2010
Resumen
Este estudio revela cómo la modificación del grupo fosfato en el fosfatidilinositol (PI) afecta su descomposición por el antibiótico Streptomyces phospholipase C (saPLC1). La sustitución de azufre en posiciones específicas altera drásticamente la actividad de las enzimas y los mecanismos de reacción.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Enzimología Enzimología.
- Biología Molecular Biología Molecular
Sus antecedentes:
- La fosfolipasa C (saPLC1) específica para el fosfatidilinositol de Streptomyces antibioticus hidroliza el fosfatidilinositol (PI) a través de un intermediario cíclico.
- Comprender el mecanismo de saPLC1 es crucial para elucidar las vías de señalización lipídica y la cinética de las enzimas.
Objetivo del estudio:
- Investigar el impacto de la sustitución de azufre en la fracción de fosfato de PI en la actividad y el mecanismo catalítico de saPLC1.
- Para analizar los productos de hidrólisis de los análogos del fosforotioato de PI y del fosforoditioato.
Principales métodos:
- Síntesis e hidrólisis enzimática de análogos de PI con azufre en las posiciones de no puente (pro-R, pro-S) y puente.
- Análisis cinético para determinar las constantes de velocidad catalítica para saPLC1 con sustratos PI modificados.
- Análisis del producto para identificar productos de hidrólisis de análogos de PI y productos intermedios cíclicos.
Principales resultados:
- La sustitución del oxígeno pro-S por azufre redujo drásticamente la actividad de saPLC1 (disminución de 3 x 10 ^ 7 veces), mientras que la sustitución pro-R tuvo un efecto menor.
- La introducción de un segundo azufre en la posición de puente del análogo de S(p) fosforotioato aumentó significativamente las tasas de escisión (2 x 10 ^ 5 veces), lo que sugiere un cambio a un mecanismo disociativo.
- La hidrólisis de los diastereómeros de fosfato cíclico (1,6-IcP) de 1,6-inositol produjo productos distintos, con la hidrólisis trans-1,6-IcP produciendo inositol 6-fosforotioato a través de la reorientación del sitio activo.
Conclusiones:
- La estereoquímica de la sustitución de azufre en los análogos de PI influye críticamente en las tasas y el mecanismo catalítico de saPLC1.
- Los análogos de S(p) fosforotioato y fosforoditioato revelan conocimientos mecanicistas sobre las etapas catalíticas de saPLC1.
- La flexibilidad del sitio activo de la enzima, demostrada por la reorientación de trans-1,6-IcP, juega un papel clave en la formación del producto.
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