6YI9

Crystal structure of the rat cytosolic PCK1, acetylated on Lys244

Summary for 6YI9

• Entry DOI: 10.2210/pdb6yi9/pdb
• Descriptor: Phosphoenolpyruvate carboxykinase, cytosolic [GTP], 1,2-ETHANEDIOL (3 entities in total)
• Functional Keywords: lyase
• Biological source: Rattus norvegicus (Norway Rat)
• Total number of polymer chains: 1
• Total formula weight: 70782.06

Authors

Latorre-Muro, P.,Baeza, J.,Hurtado-Guerrero, R.,Hicks, T.,Delso, I.,Hernandez-Ruiz, C.,Velazquez-Campoy, A.,Lawton, A.J.,Angulo, J.,Denu, J.M.,Carrodeguas, J.A. (deposition date: 2020-04-01, release date: 2020-12-23, Last modification date: 2024-10-16)

Primary citation

Latorre-Muro, P.,Baeza, J.,Hurtado-Guerrero, R.,Hicks, T.,Delso, I.,Hernandez-Ruiz, C.,Velazquez-Campoy, A.,Lawton, A.J.,Angulo, J.,Denu, J.M.,Carrodeguas, J.A.
Self-acetylation at the active site of phosphoenolpyruvate carboxykinase (PCK1) controls enzyme activity.
J.Biol.Chem., 296:100205-100205, 2021

PubMed Abstract: 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 k without changes in K 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.

PubMed: 33334880
DOI: 10.1074/jbc.RA120.015103

Experimental method

X-RAY DIFFRACTION (1.75 Å)