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Nucleoside Triphosphates - From Synthesis to Biochemical Characterization
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Cofactores sintéticos emisores: un análogo isomórfico, isofuncional y sensible del NAD
Alexander R Rovira1, Andrea Fin1, Yitzhak Tor1
1Department of Chemistry and Biochemistry, University of California, San Diego , La Jolla, California 92093-0358, United States.
Journal of the American Chemical Society
|October 19, 2017
Resumen
Se sintetizó un nuevo análogo fluorescente de NAD+, NtzAD+, y se demostró que era un sustrato viable para las enzimas clave. Su fluorescencia cambia durante las reacciones enzimáticas, lo que permite un monitoreo en tiempo real a diferencia del NAD+ nativo.
Área de la Ciencia:
- La bioquímica
- Biología Química
- Biofísica y Química
Sus antecedentes:
- El nicotinamida adenina dinucleótido (NAD+) es un cofactor crucial en numerosos procesos metabólicos.
- El monitoreo de las reacciones enzimáticas dependientes de NAD+ en tiempo real es un desafío debido a la naturaleza no fluorescente de NAD+ y NADH.
Objetivo del estudio:
- Para sintetizar y caracterizar un análogo fluorescente de NAD+, NtzAD+, para aplicaciones bioquímicas.
- Evaluar la utilidad de NtzAD+ como sustrato en reacciones enzimáticas y evaluar sus propiedades fotofísicas.
- Para demostrar el monitoreo en tiempo real de las transformaciones enzimáticas que involucran NtzAD+ utilizando espectroscopia de fluorescencia.
Principales métodos:
- Síntesis del análogo fluorescente de NAD+ basado en el núcleo de isotiazol[4,3-d]pirimidina (NtzAD+).
- Ensayos enzimáticos utilizando alcohol deshidrogenasa de levadura y lactato deshidrogenasa con NtzAD+.
- Caracterización fotofísica de NtzAD+ y su forma reducida (NtzADH).
- Evaluación del sustrato para la NADasa y las ribosiltransferasas (ART5, CTA).
Principales resultados:
- NtzAD+ y NtzADH funcionan como sustratos para las deshidrogenasas de alcohol y lactato con tasas de reacción comparables a las NAD+/NADH nativas.
- Una disminución en la fluorescencia acompaña a la conversión de NtzAD+ a NtzADH, reflejando el comportamiento de NAD+/NADH.
- NtzAD+ es procesado por NADasa y ribosil transferasas (ART5, CTA), dando lugar a la tzADP-ribosa.
- Las reacciones enzimáticas que involucran a NtzAD+ son fácilmente monitoreadas por espectroscopia de fluorescencia.
Conclusiones:
- NtzAD+ es un análogo fluorescente funcional del NAD+ adecuado para estudios bioquímicos.
- El comportamiento fotofísico complementario de NtzAD+/NtzADH permite el monitoreo de fluorescencia en tiempo real de las reacciones enzimáticas.
- NtzAD+ ofrece una ventaja significativa sobre el NAD+ no fluorescente para el estudio de enzimas y vías dependientes del NAD+.
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