Kinetic mechanism of acetyl-CoA synthase: Steady-state synthesis at variable CO/CO2 pressures

Steady-state initial rates of acetyl-CoA synthesis (v/[E-tot]) catalyzed by acetyl-CoA synthase from Clostridium thermoaceticum (ACS) were determined at various partial pressures of CO and CO2. When [CO] was Varied from 0 to 100 muM in a balance of Ar, rates increased sharply from 0.3 to 100 min(-1) . At [CO] > 100 muM, rates declined sharply and eventually stabilized at 10 min(-1) at 980 muM CO. Equivalent experiments carried out in CO2 revealed similar inhibitory behavior and residual activity under saturating [CO]. Pl ots of v/[E-tot] vs [CO2] at different fixed inhibitory [CO] revealed that V-max/[E-tot] (k(cat)) decreased with increasing [CO]. Plots of v/[E-tot] v s [CO2] at different fixed noninhibitory [CO] showed that V-max/[E-tot] was insensitive to changes in [CO]. Of eleven candidate mechanisms, the simple st one that fit the data best had the following key features: (a) either CO or CO2 (at a designated reductant level and pH) activate the enzyme (E ' CO reversible arrow E, E ' + CO2/2e(-)/2H(+) reversible arrow E); (b) CO a nd CO2 are both substrates that compete for the same enzyme form (E + CO re versible arrow ECO, E + CO2/2e(-)/2H(+) reversible arrow ECO, and ECO --> E + P); (c) between 3 and 5 molecules of CO bind cooperatively to an enzyme form different from that to which CO2 and substrate CO bind (nCO + ECO reve rsible arrow (CO),ECO), and this inhibits catalysis; and (d) the residual a ctivity arises from either the (CO),ECO state or a heterogeneous form of th e enzyme. Implications of these results, focusing on the roles of CO and CO 2 in catalysis, are discussed.