Reaction mechanism and structural model of ADP -forming acetyl-coa synthetase from the hyperthermoph
March 29th, 2008 | by admin |Reaction mechanism and structural model of ADP -forming acetyl-coa synthetase from the hyperthermophilic archaeon pyrococcus furiosus: Evidence for a second active site histidine residue.
In archaea, acetate formation and ATP synthesis from acetyl-CoA is catalysed by an unusual ADP-forming acetyl-CoA synthetase (ACD) catalyzing the formation of acetate from acetyl-CoA and concomitant ATP synthesis via substrate level phosphorylation. Together with succinyl-CoA synthetases (SCSs), ACD belongs to the protein superfamily of NDP-forming acyl-CoA synthetases. Due to sequence conservation of ACD and SCS a similar reaction mechanism was proposed including transient phosphorylation of a conserved histidine residue in the alpha subunit of both proteins. However, ACD differs from SCS in domain organisation of the subunits and in the presence of a second highly conserved histidine residue in the beta subunit, which is absent in SCS. The influence of these differences on structure and reaction mechanism of ACD was studied with heterotetrameric ACD from Pyrococcus furiosus. A structural model of P. furiosus ACD was constructed based on crystal structures of homologous proteins from Pyrococcus horikoshii. The model suggests a novel spatial arrangement of the subunits different from SCS, however, maintaining a similar catalytic site. Further, kinetic and molecular properties, enzyme phosphorylation and arsenolysis of acetyl-CoA were studied in wild type and mutant ACDs. The data indicate that ACD reaction follows a four step mechanism which differs from SCS by transient phosphorylation of a second active site histidine in the beta subunit during transfer of the phosphoryl moiety from His257alpha to ADP. (1) E + acetyl-CoA + Pi Eacetyl~P + CoA (2) Eacetyl~P acetate + E-His257alpha~P (3) E-His257alpha~P E-His71beta~P (4) E-His71beta~P + ADP ATP + E.
Bräsen C, Schmidt M, Grötzinger J, Schönheit P.
Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, Kiel, Schleswig-Holstein D-24118.