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La conversión de la tripsina en quimotripsina: el papel de los bucles de superficie
L Hedstrom1, L Szilagyi, W J Rutter
1Hormone Research Institute, University of California, San Francisco 94143-0534.
Science (New York, N.Y.)
|March 6, 1992
Resumen
Alteración de la tripsina por alteración de la misma.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Enzimología Enzimología.
- Ingeniería de proteínas Ingeniería de proteínas.
Sus antecedentes:
- La tripsina y la quimotripsina son proteasas serinas con estructuras similares pero distintas especificidades de sustrato.
- La tripsina se divide después de los residuos básicos (Arg, Lys), mientras que la quimotripsina se dirige a los grandes residuos hidrofóbicos.
- Comprender estas diferencias de especificidad puede informar a la ingeniería enzimática para aplicaciones específicas.
Objetivo del estudio:
- Investigar los determinantes estructurales de la especificidad del sustrato en la tripsina y la quimotripsina.
- Para diseñar un mutante de tripsina con una especificidad de sustrato similar a la quimotripsina.
- Aclarar las funciones del sitio de unión S1 y los bucles superficiales en la catálisis y discriminación de enzimas.
Principales métodos:
- Se utilizó la mutagenesis dirigida al sitio para reemplazar el sitio de unión S1 de la tripsina con residuos de quimotripsina.
- Otras modificaciones implicaron el intercambio de bucles superficiales (residuos 185-188 y 221-225) entre la tripsina y la quimotripsina.
- La cinética de la enzima y los ensayos de unión al sustrato se realizaron en enzimas de tipo salvaje y mutantes.
Principales resultados:
- Sustituir el sitio S1 solo transfirió la especificidad de la esterasa pero no la especificidad de la hidrólisis de la amida.
- La mutación tanto en el sitio S1 como en bucles superficiales específicos convirtió la tripsina en una enzima similar a la quimotripsina.
- El mutante diseñado exhibió la velocidad catalítica de la quimotripsina, pero tenía un deterioro de la unión al sustrato.
- La discriminación del sustrato en el mutante se produjo durante la acilación, no la unión, similar a la quimotripsina.
Conclusiones:
- Los bucles superficiales, que no forman directamente parte del sitio activo, influyen significativamente en la especificidad del sustrato de la proteasa.
- La especificidad enzimática es una interacción compleja entre el sitio activo y los elementos estructurales remotos.
- Este estudio proporciona información sobre la ingeniería de proteínas para crear enzimas con nuevas propiedades catalíticas.
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