Redox-Dependent Acetyl Transfer Partial Reaction of the Acetyl-CoA Decarbonylase/Synthase Complex:  Kinetics and Mechanism

Abstract

Acetyl-CoA decarbonylase/synthase (ACDS) is a multienzyme complex that plays a central role in energy metabolism in Methanosarcina barkeri grown on acetate. The ACDS complex carries out an unusual reaction involving net cleavage of the acetyl C-C and thioester bonds of acetyl-CoA. The overall reaction is composed of several partial reactions, one of which involves catalysis of acetyl group transfer. To gain insight into the overall reaction, a study was carried out on the kinetics and mechanism of the acetyltransferase partial reaction. Analysis by HPLC was used to quantify rates of acetyl transfer from acetyl-CoA both to 3'-dephospho-CoA and, by isotope exchange, to 14C-labeled CoA. Acetyl transfer activity was observed only under strongly reducing conditions, and was half-maximal at -486 mV at pH 6.5. The midpoint activation potential became increasingly more negative as the pH was increased, indicating the involvement of a protonation step. Cooperative dependence on acetyl-CoA concentration was exhibited in reactions that contained incompletely reduced enzyme; however, under redox conditions supporting maximum activity, hyperbolic kinetics were found. A ping-pong steady state kinetic mechanism was established, consistent with formation of an acetyl-enzyme intermediate. Analysis of the inhibitory effects of CoA on acetyl transfer to 3'-dephospho-CoA provided values for KiCoA of 6.8 microM and for Kiacetyl-CoA of 45 microM; isotope exchange analyses yielded values of 32 and 120 microM, respectively. Two separate measures of stability yielded values for the free energy of hydrolysis of the acetyl-enzyme intermediate of -9.6 and -9.3 kcal/mol, an indication of a high-energy bonding interaction in the acetyl-enzyme species. Implications for the mechanism of C-C bond cleavage are discussed.

Nickel in Subunit β of the Acetyl-CoA Decarbonylase/Synthase Multienzyme Complex in Methanogens

“…Reactions were carried out in 12 ϫ 75-mm glass tubes at 25°C and were initiated by addition of a 10-l aliquot of sufficiently diluted protein to 110 l of an assay solution that contained all the above components at concentrations needed to give the final values indicated in a total reaction volume of 120 l. A series of reactions were carried out for each enzymatic rate determination and stopped at different time points (from 0 to 8 min) by addition of 120 l of 2 mM TiCl 3 in 0.5 M sodium citrate, pH 4.0, and the mixtures were frozen in liquid nitrogen prior to analysis by HPLC for the products CoA and S-acetyl-3Ј-dephospho-CoA. HPLC analyses and calculation of reaction rates were performed as described previously (3).…”

“…One of these was the full-length 472-amino acid protein, CdhC, and the other was a 397-amino acid form truncated at the C terminus, CdhC*. CdhC* includes all major regions of conservation among ␤ subunit homologs and is about 30 amino acids smaller than the estimated size of the native protein isolated in truncated form following disruption of the ACDS complex by partial proteolysis (2,3). SDS-gel electrophoresis showed that similar amounts of CdhC and CdhC* were produced over time after induction of E. coli cultures grown under anaerobic conditions, as described under "Materials and Methods."…”

“…When reacted with CO in the presence of a reducing agent, the isolated clostridial ␣ subunit exhibited an EPR spectrum similar to the A cluster NiFeC signal found in earlier studies on the intact ACDS complex and the native clostridial CODH/ACS enzyme (11). Characterization of the ability of the ACDS ␤ subunit to bind acetyl-CoA and CoA, and to catalyze acetyl group transfer reactions by way of a high energy acetyl-enzyme intermediate formed on the enzyme at low redox potentials (3), and other studies (4), further implicated the ␤ subunit A center as the site where acetyl C-C bond activation takes place. Functional preparations of the clostridial ␣ subunit have now been obtained that catalyze acetyl-CoA formation from CoA and CO in the presence of methylated clostridial corrinoid iron-sulfur protein (12).…”

“…The ACDS complex catalyzes cleavage of the acetyl C-C bond using the substrates acetylCoA and tetrahydrosarcinapterin (H 4 SPt), a tetrahydrofolate analog which serves as methyl acceptor, and yields the products CoA, N 5 -methyltetrahydrosarcinapterin, CO 2 , and two reducing equivalents, as given in Reaction 1 (1 This overall reaction is made up of a series of partial reactions catalyzed by different protein subcomponents of the ACDS complex as shown in Scheme 1 (2). Acetyl-CoA binds to the ␤ subunit, and under low redox potential conditions, as required for activity, transfers the acetyl group to a nucleophilic center on the enzyme forming an acetyl-enzyme species and releasing CoA (Scheme 1, acetyl transfer) (2,3). The acetyl intermediate then undergoes C-C bond cleavage by a reaction that is presumed to involve metal-based decarbonylation and/or methyl group migration (Scheme 1, cleavage).…”

“…The carbonyl group is oxidized to CO 2 by a process involving the ␣⑀ CO dehydrogenase subcomponent, with regeneration of the reduced form of the ␤ subunit. Previous studies on the ␤ subunit have focused on a C-terminally truncated form of the protein purified from the native ACDS complex following partial proteolytic digestion (2)(3)(4).…”