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Síntase abietadieno bifuncional: transferencia difusiva libre del intermedio difosfato de (+) -copalilo entre dos
R J Peters1, M M Ravn, R M Coates
1Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA.
Journal of the American Chemical Society
|September 13, 2001
Resumen
La Abietadieno Sintasa (AS) utiliza dos sitios activos distintos para convertir el difosfato de geranilgeranilo en diterpenos. Estos sitios, ubicados en dominios separados, facilitan un mecanismo de difusión libre para el intermediario difosfato de copalilo.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Enzimología Enzimología.
- Ciencias de las plantas Ciencias de las plantas.
Sus antecedentes:
- La Abietadieno Sintasa (AS) inicia la biosíntesis del ácido de resina en el abeto grande (Abies grandis).
- AS cataliza dos reacciones de ciclización distintas desde el difosfato de geranilgeranil (GGPP).
- La primera reacción da como resultado (+) -copalil difosfato (CPP), un intermediario bicíclico estable.
Objetivo del estudio:
- Investigar el mecanismo catalítico y la organización del sitio activo de la Abietadieno Sintasa (AS).
- Para determinar si AS posee sitios activos distintos para sus reacciones de ciclización secuenciales.
- Para aclarar el mecanismo de transferencia intermedia entre sitios activos.
Principales métodos:
- Utilizó un inhibidor análogo de estado de transición (14,15-dihidro-15-azageranylgeranyl difosfato).
- Mutantes generados dirigidos al sitio que se dirigen a los motivos ricos en aspartato conservados.
- Realizó estudios de cinética enzimática y de complementación.
Principales resultados:
- Se demostró que AS emplea dos sitios activos distintos para ciclizaciones secuenciales.
- Las mutaciones en motivos específicos abolieron una actividad catalítica, sugiriendo dominios separados.
- La complementación de mutantes y la detección de CPP libre confirmó un mecanismo de transferencia de difusión libre.
- Las enzimas mutantes aumentaron la actividad del SA de tipo salvaje, lo que indica una interacción cinética compleja.
Conclusiones:
- Los sistemas auxiliares funcionan a través de dos sitios activos separados espacialmente dentro de dominios distintos.
- El intermedio (+) -copalil difosfato se difunde libremente entre estos sitios activos.
- Este mecanismo proporciona información sobre la complejidad cinética de la biosíntesis del diterpeno.
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