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 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.
Highlights
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
The controls associated with this experiment revealed that almost 25% of PCK1 was acetylated by acetylCoA alone when compared with PCK1 incubated in the presence of p300 and acetyl-coenzyme A (CoA) (Fig. 1A)
We observed that PCK1 acetylated with acetyl-CoA alone or together with p300 had decreased activity in both reactions under Vmax conditions compared with controls (Fig. 1C)
Summary
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). Given the ability of acetyl-CoA to directly react with lysines inside the active site of PCK1, we wondered whether acetylation was affected when PCK1 was incubated with 1 mM acetylCoA plus one of its specific substrates (OAA, GTP, PEP, or GDP) at 1 mM final concentration.
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