Redox-dependent acetyl transfer partial reaction of the acetyl-CoA decarbonylase/synthase complex: kinetics and mechanism.

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.