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

Abstract

Steady-state initial rates of acetyl-CoA synthesis (upsilon/[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 microM in a balance of Ar, rates increased sharply from 0.3 to 100 min(-1). At [CO] > 100 microM, rates declined sharply and eventually stabilized at 10 min(-1) at 980 microM CO. Equivalent experiments carried out in CO2 revealed similar inhibitory behavior and residual activity under saturating [CO]. Plots of upsilon/[E(tot)] vs [CO2] at different fixed inhibitory [CO] revealed that Vmax/[E(tot)] (kcat) decreased with increasing [CO]. Plots of upsilon/[E(tot)] vs [CO2] at different fixed noninhibitory [CO] showed that Vmax/[E(tot)] 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 right arrow over left arrow E, E' + CO2/2e-/2H+ right arrow over left arrow E); (b) CO and CO2 are both substrates that compete for the same enzyme form (E + CO right arrow over left arrow ECO, E + CO2/2e-/2H+ right arrow over left 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 right arrow over left arrow (CO)nECO), and this inhibits catalysis; and (d) the residual activity arises from either the (CO)nECO state or a heterogeneous form of the enzyme. Implications of these results, focusing on the roles of CO and CO2 in catalysis, are discussed.

Kinetics of CO conversion into H2 by Carboxydothermus hydrogenoformans

“…When both m and n constants are greater than zero, this represents generalized uncompetitive inhibition. This is consistent with the conclusion of Maynard et al (2001) that the CO inhibition of ACS purified from Clostridium thermoaceticum was uncompetitive.…”

“…The CO toxicity is not only the result of hydrogenase inhibition that may occur even at CO partial pressure (p CO ) as low as 0.05 atm (≈45 μM CO in solution, Soboh et al 2002); CO inhibits also the acetyl-CoA synthesis, usually at higher concentrations. But typically a residual activity remains at the highest CO concentrations employed (Maynard et al 2001;Amos 2004). Complete inhibition of direct conversion of CO into CH 4 and CO 2 by Methanobacterium thermoautotrophicum occurred at a p CO of 0.6 atm (Daniels et al 1977), whereas Methanosarcina barkeri could be slowly adapted to growth at 100% CO (O'Brien et al 1984).…”

Potential of Wastewater-Treating Anaerobic Granules for Biomethanation of Synthesis Gas

“…The CO-activity profile exhibited a close similarity with that of enzymatic acetyl-CoA synthesis rates as measured at various CO concentrations by Maynard and Coll, 25 with a typical residual steady activity for the same CO range. This residual activity of the acetyl-CoA-synthase (ACS) along with the high CO-sensitivity of hydrogenases 16 likely explained the accumulation of methane precursors, predominantly acetate, at higher CO concentrations ( Figure 2B).…”

Pulse-Chase Studies of the Synthesis of Acetyl-CoA by Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase

“…That methylation of CODH⅐ACS or recombinant (ACS HT ) (31,32) by CH 3 -CFeSP occurs faster than acetyl-CoA synthesis also suggests that methylation is the first step in the synthesis (33). Furthermore, CO acts as a substrate inhibitor of acetyl-CoA synthesis (34,35). On the other hand, acetyl-CoA synthesis is linearly dependent on CO at concentrations likely to be physiologically relevant, with CO becoming inhibitory only at relatively high concentrations (Ͼ100 M) (34).…”

“…Furthermore, CO acts as a substrate inhibitor of acetyl-CoA synthesis (34,35). On the other hand, acetyl-CoA synthesis is linearly dependent on CO at concentrations likely to be physiologically relevant, with CO becoming inhibitory only at relatively high concentrations (Ͼ100 M) (34). Furthermore, CODH⅐ACS can be carbonylated at catalytically relevant rates to form a paramagnetic adduct called the NiFeC species (17,36), and the carbonylated enzyme can then undergo methylation and CoA binding to form acetyl-CoA at catalytically relevant rates (33).…”