Characterization of acetyl-CoA synthetase kinetics and ATP-binding

Abstract

BackgroundThe superfamily of adenylating enzymes is a large family of enzymes broadly distributed from bacteria to humans. Acetyl-CoA synthetase (Acs), member of this family, is a metabolic enzyme with an essential role in Escherichia coli (E. coli) acetate metabolism, whose catalytic activity is regulated by acetylation/deacetylation in vivo. MethodsIn this study, the kinetics and thermodynamic parameters of deacetylated and acetylated E. coli Acs were studied for the adenylating step. Moreover, the role of the T264, K270, D500 and K609 residues in catalysis and ATP-binding was also determined by Isothermal titration calorimetry. ResultsThe results showed that native Acs enzyme binds ATP in an endothermic way. The dissociation constant has been determined and ATP-binding showed no significant differences between acetylated and deacetylated enzyme, although kcat was much higher for the deacetylated enzyme. However, K609 lysine mutation resulted in an increase in ATP-Acs-affinity and in a total loss of enzymatic activity, while T264 and D500 mutant proteins showed a total loss of ATP-binding ability and a decrease in catalytic activity. K609 site-specified acetylation induced a change in Acs conformation which resulted in an exothermic and more energetic ATP-binding. ConclusionsThe differences in ATP-binding could explain the broadly conserved inactivation of Acs when K609 is acetylated. General SignificanceThe results presented in this study demonstrate the importance of the selected residues in Acs ATP-binding and represent an advance in our understanding of the adenylation step of the superfamily of adenylating enzymes and of their acetylation/deacetylation regulation.