Kinetic Mechanism of Acetyl-CoA Synthase:  Steady-State Synthesis at Variable CO/CO2 Pressures

Steady-state initial rates of acetyl-CoA synthesis (υ/[Etot]) 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 μM in a balance of Ar, rates increased sharply from 0.3 to 100 min-1. At [CO] > 100 μM, rates declined sharply and eventually stabilized at 10 min-1 at 980 μM CO. Equivalent experiments carried out in CO2 revealed similar inhibitory behavior and residual activity under saturating [CO]. Plots of υ/[Etot] vs [CO2] at different fixed inhibitory [CO] revealed that V max/[Etot] (k cat) decreased with increasing [CO]. Plots of υ/[Etot] vs [CO2] at different fixed noninhibitory [CO] showed that V max/[Etot] was insensitive to changes in [CO]. Of eleven candidate mechanisms, the simplest 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 ⇄ E, E‘ + CO2/2e-/2H+ ⇄ E); (b) CO and CO2 are both substrates that compete for the same enzyme form (E + CO ⇄ ECO, E + CO2/2e-/2H+ ⇄ 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 ⇄ (CO) n ECO), and this inhibits catalysis; and (d) the residual activity arises from either the (CO) n ECO state or a heterogeneous form of the enzyme. Implications of these results, focusing on the roles of CO and CO2 in catalysis, are discussed.