Reaction Mechanism and Structural Model of ADP-forming Acetyl-CoA Synthetase from the Hyperthermophilic Archaeon Pyrococcus furiosus

In Archaea, acetate formation and ATP synthesis from acetyl-CoA is catalyzed by an unusual ADP-forming acetyl-CoA synthetase (ACD) (acetyl-CoA + ADP + P_i ⇆ acetate + ATP + HS-CoA) catalyzing the formation of acetate from acetyl-CoA and concomitant ATP synthesis by the mechanism of substrate level phosphorylation. ACD belongs to the protein superfamily of nucleoside diphosphate-forming acyl-CoA synthetases, which also include succinyl-CoA synthetases (SCSs). ACD differs from SCS in domain organization of subunits and in the presence of a second highly conserved histidine residue in the β-subunit, which is absent in SCS. The influence of these differences on structure and reaction mechanism of ACD was studied with heterotetrameric ACD (α₂β₂) from the hyperthermophilic archaeon Pyrococcus furiosus in comparison with heterotetrameric SCS. A structural model of P. furiosus ACD was constructed suggesting a novel spatial arrangement of the subunits different from SCS, however, maintaining a similar catalytic site. Furthermore, kinetic and molecular properties and enzyme phosphorylation as well as the ability to catalyze arsenolysis of acetyl-CoA were studied in wild type ACD and several mutant enzymes. The data indicate that the formation of enzyme-bound acetyl phosphate and enzyme phosphorylation at His-257α, respectively, proceed in analogy to SCS. In contrast to SCS, in ACD the phosphoryl group is transferred from the His-257α to ADP via transient phosphorylation of a second conserved histidine residue in theβ-subunit, His-71β. It is proposed that ACD reaction follows a novel four-step mechanism including transient phosphorylation of two active site histidine residues: