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Estudios mecánicos sobre la tiamina pirimidina sintasa de levadura de una sola vuelta: Caracterización de la enzima

Rung-Yi Lai¹, Anushree Mondal¹, Dmytro Fedoseyenko¹

¹Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.

Journal of the American Chemical Society

|June 8, 2022

Resumen

La tiamina pirimidina sintasa eucariota (THI5p) convierte su sitio activo de histidina y fosfato piridoxal (PLP) en tiamina pirimidina (HMP-P). Los investigadores identificaron fragmentos clave y el cofactor de hierro, proponiendo un mecanismo de reacción.

Área de la Ciencia:

La bioquímica
Enzimología
Proteomía

Sus antecedentes:

La tiamina pirimidina sintasa (THI5p) es una enzima eucariota crucial para la biosíntesis de la tiamina.
THI5p funciona como una enzima suicida / de un solo giro, convirtiendo los residuos del sitio activo y el fosfato piridoxal (PLP) en la fracción de tiamina pirimidina (HMP-P).
El mecanismo preciso y los intermediarios involucrados en esta reacción enzimática única siguen siendo incompletamente entendidos.

Objetivo del estudio:

Para aclarar el mecanismo de reacción de la tiamina pirimidina sintasa eucariota (THI5p).
Identificar los fragmentos específicos de histidina y fosfato piridoxal (PLP) generados durante la reacción catalizada por THI5p.
Caracterizar la forma activa del cofactor hierro y determinar la dependencia de oxígeno de la reacción THI5p.

Principales métodos:

Se empleó la proteómica de abajo hacia arriba para analizar el sitio activo de la enzima.
Se utilizó la espectrometría de masas por cromatografía líquida (LC-MS) para la identificación de fragmentos.
Se utilizaron métodos espectroscópicos para identificar el cofactor hierro y cuantificar los requerimientos de oxígeno.

Principales resultados:

El estudio identificó con éxito los fragmentos de histidina y fosfato piridoxal (PLP) resultantes de la reacción THI5p.
Se identificó la forma activa del cofactor de hierro implicado en el proceso catalítico.
Se determinó cuantitativamente el requerimiento de oxígeno para la conversión catalizada por THI5p.

Conclusiones:

Los fragmentos identificados y el cofactor proporcionan información crítica sobre la vía de reacción THI5p.
Se ha formulado una propuesta mecanicista para la reacción suicida/de giro único de THI5p basada en los hallazgos experimentales.
Esta investigación avanza en la comprensión de la biosíntesis de tiamina y los mecanismos enzimáticos que involucran a las enzimas dependientes de PLP.

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