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Incorporación de fluorotirosinas en la ribonucleotide reductasa utilizando una aminoacil-tRNA sintetasa
Ellen C Minnihan1, Douglas D Young, Peter G Schultz
1Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
Journal of the American Chemical Society
|September 15, 2011
Resumen
Los investigadores diseñaron enzimas para incorporar fluorotirosinas en una enzima clave, revelando nuevos conocimientos sobre los mecanismos radicales en la catálisis biológica. Este avance ayuda a estudiar el salto radical y la actividad enzimática.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Ingeniería de enzimas Ingeniería de enzimas Ingeniería de enzimas
- Química biofísica y bioquímica.
Sus antecedentes:
- Los radicales tirosílicos (Y·s) son cruciales en la catálisis biológica, formados por la pérdida de protones y electrones.
- Las fluorotirosinas (F ((n) Ys) ofrecen propiedades sintonizables para el estudio de los mecanismos de Y.
- La ribonucleótido reductasa (RNR) utiliza Y·s para el salto radical durante la catálisis.
Objetivo del estudio:
- Para desarrollar las aminoacil-tRNA sintetasas (aaRSs) para la incorporación de 2,3,5-trifluorotirosina (2,3,5-F(3) Y).
- Para incorporar específicamente en el sitio varias F ((n) Ys en Escherichia coli clase Ia RNR.
- Investigar el impacto de la incorporación de F (n) Y en la actividad de RNR y los mecanismos radicales.
Principales métodos:
- Evolución dirigida de los aaRS para el cobro F{\displaystyle F} n) Y.
- Incorporación específica del sitio de F (n) Y en las subunidades RNR (α2 y β2).
- Espectroscopia de resonancia paramagnética de electrones (EPR) para caracterizar los radicales F (n) Y.
Principales resultados:
- Los aaRSs diseñados cargaron específicamente los tRNAs ortogonales con F ((n) Ys sobre la tirosina.
- La incorporación de F(n) Ys en RNR, incluso en el sitio Y· (β2, posición 122), produjo proteínas funcionales.
- Se generaron radicales F ((n) Y de larga vida en la posición 122, lo que permitió la caracterización de la EPR.
- El mutante Y ((122) ((2,3,5) F ((3)) Y-β2 mantuvo la actividad pero permitió la acumulación de un nuevo radical en la vía.
Conclusiones:
- La evolución dirigida produjo con éxito aaRSs para la incorporación de F.
- La sustitución F (n) Y en RNR proporciona una herramienta para estudiar las vías radicales.
- Las distintas señales EPR de los radicales F (n) Y facilitan la identificación de nuevos intermediarios.
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