130479 20240125162931.0 doi 10.1074/jbc.RA120.015103 sideral 122576 ART-2021-122576 eng Latorre-Muro, Pedro (orcid)0000-0002-6794-3535 Self-acetylation at the active site of phosphoenolpyruvate carboxykinase (PCK1) controls enzyme activity 2021 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. Access copy available to the general public Unrestricted info:eu-repo/grantAgreement/ES/DGA/E45-17R info:eu-repo/grantAgreement/ES/MEC/AGL2015–66177-R info:eu-repo/grantAgreement/ES/UZ/2015-BIO-01 info:eu-repo/semantics/openAccess by http://creativecommons.org/licenses/by/3.0/es/ 5.485 2021 BIOCHEMISTRY & MOLECULAR BIOLOGY 94 / 297 = 0.316 2021 Q2 T1 1.871 2021 Cell Biology 2021 Q1 Biochemistry 2021 Q1 8.8 2021 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion Baeza, Josué Hurtado-Guerrero, Ramón (orcid)0000-0002-3122-9401 Hicks, Thomas Delso, Ignacio Universidad de Zaragoza (orcid)0000-0001-8355-2289 Hernández-Ruiz, Cristina Velazquez-Campoy, Adrián Universidad de Zaragoza (orcid)0000-0001-5702-4538 Lawton, Alexis J. Angulo, Jesús Denu, John M. Carrodeguas, José A. Universidad de Zaragoza (orcid)0000-0003-0062-1029 2013 765 Universidad de Zaragoza Dpto. Química Orgánica Área Química Orgánica 1002 060 Universidad de Zaragoza Dpto. Bioq.Biolog.Mol. Celular Área Bioquímica y Biolog.Mole. 296 (2021), 100205 [13 pp.] J. biol. chem. Journal of Biological Chemistry 0021-9258 3228615 http://zaguan.unizar.es/record/130479/files/texto_completo.pdf Versión publicada 3602897 http://zaguan.unizar.es/record/130479/files/texto_completo.jpg?subformat=icon icon Versión publicada oai:zaguan.unizar.es:130479 articulos driver 2024-01-25-15:14:01 ARTICLE