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

Latorre-Muro, Pedro, Baeza, Josue, Hurtado-Guerrero, Ramon, Hicks, Thomas, Delso, Ignacio ORCID:, Hernández-Ruiz, Cristina, Velázquez-Campoy, Adrian, Lawton, Alexis J., Angulo, Jesús ORCID:, Denu, John M. and Carrodeguas, José A. (2021) Self-acetylation at the active site of phosphoenolpyruvate carboxykinase (PCK1) controls enzyme activity. Journal of Biological Chemistry, 296. ISSN 0021-9258

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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, saturation-transfer 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.

Item Type: Article
Additional Information: Data availability statement: All data are contained within the article. Besides, the PDB code for our structure has also been deposited at the PDB server (see the link The mass spectrometry raw files and the result files used in this study have been deposited to the ProteomeXchange Consortium via the MassIVE partner repository and can be accessed through either the ProteomExchange dataset identifier PXD021745 or MassIVE ID MSV000086210. URL:; MassIVE ID: MSV000086210; ProteomeXchange ID: PXD021745.
Uncontrolled Keywords: biochemistry,molecular biology,cell biology ,/dk/atira/pure/subjectarea/asjc/1300/1303
Faculty \ School: Faculty of Science > School of Pharmacy
UEA Research Groups: Faculty of Science > Research Groups > Pharmaceutical Materials and Soft Matter
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Depositing User: LivePure Connector
Date Deposited: 24 May 2022 14:59
Last Modified: 08 Mar 2024 22:31
DOI: 10.1074/jbc.RA120.015103


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