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Probeando la catálisis ácida general en la ribozima cabeza de martillo
Jason M Thomas1, David M Perrin
1Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, B.C., Canada, V6T 1Z1.
Journal of the American Chemical Society
|January 22, 2009
Resumen
Los cationes metálicos divalentes (M(2+)) y un grupo específico de ribozima son cruciales para la catálisis ácida general de la ribozima cabeza de martillo. Este estudio aclara sus funciones, mostrando que la coordinación del cofactor M2+ reduce el G8 2'-OH pK (a).
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Biología Molecular Biología Molecular
- Enzimología Enzimología.
Sus antecedentes:
- Las ribozimas de cabeza de martillo son enzimas de ARN con un núcleo catalítico.
- Estudios recientes sugieren que grupos específicos de ribozimas y iones metálicos están involucrados en la catálisis.
- El papel catalítico preciso de los cationes metálicos divalentes (M(2+)) en las ribozimas de cabeza de martillo sigue sin estar claro.
Objetivo del estudio:
- Para caracterizar funcionalmente el mecanismo general de catálisis ácida en el S. mansoni ribozima de cabeza de martillo.
- Para aclarar el papel de los cationes metálicos divalentes (M(2+)) como cofactores en este proceso catalítico.
- Investigar la contribución del residuo de ribozima G8 2 extquotesingle-OH al catálisis ácida general.
Principales métodos:
- Análisis comparativo de la escisión de la ribozima de la cabeza de martillo en los sustratos naturales de ribofosfodiéster y puente-5 extquotesingle-phosphorothioate.
- Mutagénesis dirigida al sitio del residuo de ribozima G8 2 extquotesingle-OH.
- Sustitución de los cationes metálicos divalentes (M(2+)) como cofactores.
- Síntesis de sustratos de puente radiomarcado-5 extquotesingle-phosphorothioate.
Principales resultados:
- La hendidura de los sustratos naturales fue inhibida por la modificación G8 2 extquotesingle-OH y la presencia/identidad del cofactor M(2+).
- La hendidura de los sustratos de fosforotioato fue insensible a estas modificaciones.
- Se observó una perturbación general del ácido pK (a) con la sustitución M2+ y la modificación G8 2 extquotesingle-OH.
Conclusiones:
- Tanto los cofactores M(2+) como el G8 2 extquotesingle-OH son esenciales para la catálisis ácida general de la ribozima cabeza de martillo.
- M(2+) no estabilizan el grupo saliente a través de las interacciones del ácido de Lewis.
- El estado de transición M(2+) coordinación de G8 2 extquotesingle-OH probablemente reduce su pK(a), mejorando la transferencia de protones al grupo saliente.
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