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Interacción de la proteína del sustrato en la triptófano dioxigenasa humana: el papel crítico de H76
Dipanwita Batabyal1, Syun-Ru Yeh
1Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, usa.
Journal of the American Chemical Society
|February 13, 2009
Resumen
La investigación de la histidina 76 (H76) en la triptófano humana 2,3-dioxigenasa (hTDO) revela su papel crucial como una base activa en el sitio. Las mutaciones H76S y H76A perjudican significativamente la actividad enzimática al interrumpir la catálisis y la unión al sustrato.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Enzimología Enzimología.
- Ciencia de las proteínas Ciencia de las proteínas.
Sus antecedentes:
- La vía de la kynurenina se inicia con la oxidación del triptófano, un paso crucial catalizado por hTDO y hIDO.
- Se supone que la histidina 76 (H76) en hTDO funciona como una base del sitio activo, esencial para la oxidación del triptófano (Trp).
- El residuo homólogo en hIDO es una serina, lo que sugiere una divergencia funcional.
Objetivo del estudio:
- Aclarar la importancia funcional y estructural del residuo H76 conservado en la triptófano humana 2,3-dioxigenasa (hTDO).
- Investigar el mecanismo catalítico y la unión al sustrato de hTDO a través de la mutagénesis dirigida en el sitio.
Principales métodos:
- Se empleó mutagenesis dirigida al sitio para crear mutantes H76S y H76A de hTDO.
- Se realizaron ensayos de actividad enzimática para determinar los parámetros cinéticos (kcat y KM).
- La espectroscopia Raman de resonancia se utilizó con análogos de triptófano para sondear las interacciones del sitio activo.
Principales resultados:
- Tanto las mutaciones H76S como las H76A dieron como resultado una disminución de la eficiencia catalítica (kcat) y un aumento de la afinidad con el sustrato (KM).
- La reducción en kcat se atribuye a la pérdida del catalizador base H76, reemplazado por especies catalíticas más débiles como el agua.
- El aumento de KM sugiere que H76 contribuye a las interacciones específicas del sustrato con las proteínas y al posicionamiento del sustrato dentro del sitio activo.
Conclusiones:
- El residuo H76 es crítico tanto para la actividad catalítica como para la unión al sustrato de hTDO.
- La mutación de H76 interrumpe la intrincada red de interacciones dentro del sitio activo, afectando la función general de la enzima.
- Estos hallazgos resaltan la importancia de H76 en la estabilización del complejo ternario activo (l-Trp-O2-hTDO).
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