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J. Biol. Chem., Vol. 283, Issue 22, 15409-15418, May 30, 2008

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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^*

Christopher Bräsen, Marcel Schmidt, Joachim Grötzinger, and Peter Schönheit¹

From the Institut für Allgemeine Mikrobiologie, Christian-Albrechts Universität Kiel and the Institut für Biochemie, Christian-Albrechts-Universität, D-24118 Kiel, Germany

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 (₂β₂) 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-257, respectively, proceed in analogy to SCS. In contrast to SCS, in ACD the phosphoryl group is transferred from the His-257 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:

Received for publication, December 14, 2007 , and in revised form, March 26, 2008.

^* This work was supported by grants of the Deutsche Forschungsgemeinschaft (SCHO 316/10-1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-3 and Table 1.

¹ To whom correspondence should be addressed: Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität, Kiel, Am Botanischen Garten 1-9, D-24118 Kiel, Germany. Tel.: 49-431-8804328; Fax: 49-8802194; E-mail: peter.schoenheit{at}ifam.uni-kiel.de.

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