Methods for analysis of acetyl-CoA synthase applications to bacterial and archaeal systems

• Published in: Methods in enzymology Vol. 494; p. 189
• Main Author: Grahame, David A
• Format: Journal Article
• Language: English
• Published: United States 2011
• Subjects: Acetate-CoA Ligase - metabolism; Archaea - enzymology; Bacteria - enzymology; Chromatography, High Pressure Liquid; Models, Biological
• ISSN: 1557-7988
• DOI: 10.1016/B978-0-12-385112-3.00010-X

The nickel- and iron-containing enzyme acetyl-CoA synthase (ACS) catalyzes de novo synthesis as well as overall cleavage of acetyl-CoA in acetogens, various other anaerobic bacteria, methanogens, and other archaea. The enzyme contains a unique active site metal cluster, designated the A cluster, that consists of a binuclear Ni-Ni center bridged to an [Fe(4)S(4)] cluster. In bacteria, ACS is tightly associated with CO dehydrogenase to form the bifunctional heterotetrameric enzyme CODH/ACS, whereas in archaea, ACS is a component of the large multienzyme complex acetyl-CoA decarbonylase/synthase (ACDS), which comprises five different subunits that make up the subcomponent proteins ACS, CODH, and a corrinoid enzyme. Characteristic properties of ACS are discussed, and key methods are described for analysis of the enzyme's multiple redox-dependent activities, including overall acetyl-CoA synthesis, acetyltransferase, and an isotopic exchange reaction between the carbonyl group of acetyl-CoA and CO. Systematic measurement of these activities, applied to different ACS protein forms, provides insight into the ACS catalytic mechanism and physiological functions in both CODH/ACS and ACDS systems.