Acetyl-coenzyme A Synthetase (Acs) Assay

Abstract

Acetyl-coenzyme A synthethase (Acs, E.C.6.2.1.1) is an acetate activating enzyme widely represented in nature from bacteria to human. Its function is important for cellular catabolism, especially in order to support microbial growth at low concentrations of acetate (et al., 2011; Castano Cerezo et al., 2009；Renilla et al., 2012). In this protocol, a continuous coupled enzymatic assay for Acs activity is described. Product formation is followed spectrophotometrically by the formation of NADH. The protocol is tailored for E. coli’s Acs, but it can be adapted to assay Acs in any other organism.The acetyl-coenzyme A synthetase (Acs) assay was first described by Brown et al. (1977). Acs activity is measured using an enzymatic method coupled to malate dehydrogenase (Mdh) and citrate synthase (Cs):(Acs) acetate + CoASH + ATP -> acetyl-CoA + AMP(Cs) acetyl-CoA + oxaloacetate -> citrate + CoASH(Mdh) L-malate + NAD+ -> oxaloacetate + NADHNet reaction: Acetate + ATP + L-malate + NAD+ -> citrate + AMP + NADHUnder the assay conditions, Mdh and Cs activities are in excess and the rate of NADH formation is limited by Acs activity.