Self-acetylation at the active site of phosphoenolpyruvate carboxykinase (PCK1) controls enzyme activity

Latorre-Muro, Pedro ; Baeza, Josué ; Hurtado-Guerrero, Ramón ; Hicks, Thomas ; Delso, Ignacio (Universidad de Zaragoza) ; Hernández-Ruiz, Cristina ; Velazquez-Campoy, Adrián (Universidad de Zaragoza) ; Lawton, Alexis J. ; Angulo, Jesús ; Denu, John M. ; Carrodeguas, José A. (Universidad de Zaragoza)

Resumen: Acetylation is known to regulate the activity of cytosolicphosphoenolpyruvate carboxykinase (PCK1), a key enzyme ingluconeogenesis, by promoting the reverse reaction of theenzyme (converting phosphoenolpyruvate to oxaloacetate). It isalso known that the histone acetyltransferase p300 can inducePCK1 acetylation in cells, but whether that is a direct or in-direct function was not known. Here we initially set out todetermine whether p300 can acetylate directly PCK1in vitro.We report that p300 weakly acetylates PCK1, but surprisingly,using several techniques including protein crystallization, massspectrometry, isothermal titration calorimetry, saturation-transfer difference nuclear magnetic resonance and moleculardocking, we found that PCK1 is also able to acetylate itselfusing acetyl-CoA independently of p300. This reaction yieldedan acetylated recombinant PCK1 with a 3-fold decrease inkcatwithout changes inKmfor all substrates. Acetylation stoichi-ometry was determined for 14 residues, including residueslining the active site. Structural and kinetic analyses deter-mined that site-directed acetylation of K244, located inside theactive site, altered this site and rendered the enzyme inactive.In addition, we found that acetyl-CoA binding to the active siteis specific and metal dependent. Ourfindings provide directevidence for acetyl-CoA binding and chemical reaction withthe active site of PCK1 and suggest a newly discovered regu-latory mechanism of PCK1 during metabolic stress.

Idioma: Inglés

DOI: 10.1074/jbc.RA120.015103

Año: 2021

Publicado en: Journal of Biological Chemistry 296 (2021), 100205 [13 pp.]
ISSN: 0021-9258

Factor impacto JCR: 5.485 (2021)

Categ. JCR: BIOCHEMISTRY & MOLECULAR BIOLOGY rank: 94 / 297 = 0.316 (2021) - Q2 - T1

Factor impacto CITESCORE: 8.8 - Biochemistry, Genetics and Molecular Biology (Q1)

Factor impacto SCIMAGO: 1.871 - Cell Biology (Q1) - Biochemistry (Q1)

Financiación: info:eu-repo/grantAgreement/ES/DGA/E45-17R
Financiación: info:eu-repo/grantAgreement/ES/MEC/AGL2015–66177-R
Financiación: info:eu-repo/grantAgreement/ES/UZ/2015-BIO-01

Tipo y forma: Artículo (Versión definitiva)

Área (Departamento): Área Química Orgánica (Dpto. Química Orgánica)
Área (Departamento): Área Bioquímica y Biolog.Mole. (Dpto. Bioq.Biolog.Mol. Celular)