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Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092
Published on: October 2, 2017
Transporte de carbamato en la carbamoil fosfato sintetasa: una investigación teórica y experimental
Liliya Lund1, Yubo Fan, Qiang Shao
1Department of Chemistry, Texas A&M University, College Station, Texas 77843-3255, USA.
Journal of the American Chemical Society
|March 2, 2010
Resumen
La carbamoil fosfato sintetasa (CPS) utiliza un túnel para transportar el carbamato dentro de la enzima. La dinámica molecular y la mutagénesis revelan barreras energéticas y bolsas de agua cruciales para este transporte, con mutaciones que reducen significativamente la actividad enzimática.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Biología Estructural Biología estructural.
- Catalización de enzimas por catálisis enzimática.
Sus antecedentes:
- La carbamoil fosfato sintetasa (CPS) cataliza un paso clave en la biosíntesis de nucleótidos y aminoácidos.
- El carbamato, un intermediario, debe ser transportado de manera eficiente dentro de la subunidad grande de la enzima desde su sitio de formación hasta su sitio de utilización.
- La comprensión de este mecanismo de transporte es crucial para elucidar la función y regulación de las enzimas.
Objetivo del estudio:
- Para investigar la vía de transporte del carbamato a través de la gran subunidad de Escherichia coli CPS.
- Identificar las principales características estructurales y paisajes energéticos que rigen la translocación del carbamato.
- Validar el papel de regiones de túnel específicas en la actividad enzimática a través de la mutagénesis.
Principales métodos:
- Se emplearon simulaciones de dinámica molecular (DM) para modelar el transporte de carbamato.
- Los cálculos de Potenciales de Fuerza Media (PMF) se utilizaron para determinar los perfiles de energía libre a lo largo del túnel de transporte.
- Se realizó mutagénesis dirigida al sitio en residuos clave dentro del túnel de carbamato propuesto.
Principales resultados:
- El túnel de transporte de carbamato consta de tres bolsas de agua y dos estrechas constricciones, creando barreras de energía libre de 6,7 y 8,4 kcal/mol.
- La translocación del carbamato es facilitada por cambios conformacionales que implican la rotación del Arg-306 al liberarse el fosfato, abriendo el túnel.
- Los mutantes A23F y G575F, diseñados para obstruir partes de túneles estrechos, exhibieron una actividad catalítica significativamente reducida (1.7% y 3.8% del tipo salvaje, respectivamente).
Conclusiones:
- El túnel de carbamato en E. coli CPS presenta importantes barreras energéticas que influyen en la eficiencia de la translocación.
- La flexibilidad conformal, particularmente la rotación del Arg-306, es esencial para abrir el túnel y facilitar el paso del carbamato.
- Los residuos específicos dentro de las partes más estrechas del túnel son críticos para mantener la actividad catalítica de CPS.
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