Artículo
Self-acetylation at the active site of phosphoenolpyruvate carboxykinase (PCK1) controls enzyme activity
Autor/es | Latorre Muro, Pedro
Baeza, Josue Hurtado Guerrero, Ramón Hicks, Thomas Delso, Ignacio Hernández Ruiz, Cristina Velázquez Campoy, Adrián Lawton, Alexis J. Angulo Álvarez, Jesús Denu, John M. Carrodeguas, José Alberto |
Departamento | Universidad de Sevilla. Departamento de Química orgánica |
Fecha de publicación | 2021 |
Fecha de depósito | 2021-04-07 |
Publicado en |
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Resumen | Acetylation is known to regulate the activity of cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme in gluconeogenesis, by promoting the reverse reaction of the enzyme (converting phosphoenolpyruvate to ... Acetylation is known to regulate the activity of cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme in gluconeogenesis, by promoting the reverse reaction of the enzyme (converting phosphoenolpyruvate to oxaloacetate). It is also known that the histone acetyltransferase p300 can induce PCK1 acetylation in cells, but whether that is a direct or indirect function was not known. Here we initially set out to determine whether p300 can acetylate directly PCK1 in vitro. We report that p300 weakly acetylates PCK1, but surprisingly, using several techniques including protein crystallization, mass spectrometry, isothermal titration calorimetry, saturationtransfer difference nuclear magnetic resonance and molecular docking, we found that PCK1 is also able to acetylate itself using acetyl-CoA independently of p300. This reaction yielded an acetylated recombinant PCK1 with a 3-fold decrease in kcat without changes in Km for all substrates. Acetylation stoichiometry was determined for 14 residues, including residues lining the active site. Structural and kinetic analyses determined that site-directed acetylation of K244, located inside the active site, altered this site and rendered the enzyme inactive. In addition, we found that acetyl-CoA binding to the active site is specific and metal dependent. Our findings provide direct evidence for acetyl-CoA binding and chemical reaction with the active site of PCK1 and suggest a newly discovered regulatory mechanism of PCK1 during metabolic stress. |
Identificador del proyecto | AGL2015-66177
UZ-2015-BIO-01 CTQ2013-44367-C2-2-P BFU2016-75633-P BFU2016-78232-P PID2019-109395GB-100 E34-R17 LMP58-18 E45-17R BioStruct-X N283570 BIOSTRUCTX-5186 BB/P010660/1 GM065386 |
Cita | Latorre Muro, P., Baeza, J., Hurtado Guerrero, R., Hicks, T., Delso, I., Hernández Ruiz, C.,...,Carrodeguas, J.A. (2021). Self-acetylation at the active site of phosphoenolpyruvate carboxykinase (PCK1) controls enzyme activity. Journal of Biological Chemistry, 296, 100205. |
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