Fig. 7: H139 is important for the Pta activity of AcsA•AcuA. | Nature Communications

Fig. 7: H139 is important for the Pta activity of AcsA•AcuA.

From: Acetyl-CoA synthetase activity is enzymatically regulated by lysine acetylation using acetyl-CoA or acetyl-phosphate as donor molecule

Fig. 7

a Main cluster of AcP within the active site of AcuA in the AcsA•AcuA complex from MD simulation (R3). R108 binds the phosphate group of AcP (RSA; RS: reaction step) and the acetyl-group is stabilized in a hydrophobic pocket. The thiol group of CoA is close to the acetyl-group C-atom of AcP (RSB), priming it for transfer of the acetyl-group. MD simulations reveal H139 of AcuA coming in close contact with the thiol of CoA, allowing to abstract a proton to activate CoA (RSC). Source data are provided as Source Data file. b Mutational studies reveal that H139 is essential for Pta activity. AcsA (20 µM) was incubated (2 h or 5 h; 37 °C) with AcuA/AcuA mutants (5 µM), AcP (1 mM), CoA (2 mM). EDTA (10 mM) was used to unravel a potential contribution of a metal ion on catalysis. The result was confirmed in at least three independent experiments. Source data are provided as Source Data file. c In the presence of AcP and CoA K549 of AcsA is acetylated within the AcsA•AcuA complex. Acetylation is increased upon mutation of E135 in AcuA and is reversed by the classical deacetylase AcuC. AcuA (1 µM) was mixed with AcsA (10 µM) in the presence of 1 mM AcP and 2 mM CoA, incubated for 3 h at 37 °C, and then, AcuC was added for 30 min at 37 °C. Acetylation of AcsA was analysed by immunoblotting (IB: AcK). Ponceau S-red staining: loading control (LC: PoS). The result was confirmed in at least three independent experiments. Source data are provided as Source Data file. d Mechanism of Pta activity via H139 acting as a general base. The results suggest H139 acting as a general base to deprotonate and thereby activate the CoA thiol group for nucleophilic attack on the electrophilic C-atom of the acetyl-group of AcP. This is enhanced by R108 of AcuA neutralizing negative charges at the AcP phosphate. A tetrahedral intermediate forms being resolved by H139 acting as general acid protonating the phosphate leaving group finally resulting in the formation of acetyl-CoA and inorganic phosphate.

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