Acetyl-CoA synthesis

The most important physiological role of CODH in the metabolism of acetogenic bacteria was unknown until 1985, when it was shown that the enzyme is bifunctional and has acetyl-CoA synthase activity (121). It was previously thought that acetyl-CoA was synthesized at the cobalt center of a vitamin-Bi2-containing protein. In the same paper, it was proposed that nickel is the active site of CO oxidation and acetyl-CoA synthesis. 

Fractionation of a five-component system from C. thermoaceticum that catalyzes acetyl-CoA synthesis. CODH activity was in fraction F3. 

CODH from acetogens found to catalyze acetyl-CoA synthesis and nickel proposed to be the active site of CO oxidation and acetyl-CoA synthesis. CODH found to contain a heterometallic cluster consisting of nickel and iron that binds CO and proposed to be the active site of acetyl-CoA synthesis. Growth of acetogens on nitrate disables the acetyl-CoA pathway. ... 

Corrinoid iron-sulfur protein (CFeSP) purified and characterized acetyl-CoA synthesis reconstituted from CH3-H4 folate, CO, and CoA using purified proteins. ... 

Wei-Ping won the bet. A series of rapid kinetic experiments provided strong support for the concept of two independent active sites. CODH/ACS was reacted with CO and the rate of development of each of the enzyme s characteristic EPR signals was compared with the rates of CO oxidation and acetyl-CoA synthesis. On the basis of these... 

CO oxidation reaction. The spectral changes in Cluster C are followed hy Cluster B reduction with a rate constant that is similar to the steady-state value. On the other hand, the rate of formation of the characteristic EPR signal for the CO adduct at Cluster A is much slower. Its rate constant matches that for acetyl-CoA synthesis, hut is several orders of magnitude slower than CO oxidation. Therefore, it was proposed that the following steps are involved in CO oxidation (1) CO hinds to Cluster C, (2) EPR spectral changes in Cluster C are accompanied hy oxidation of CO to CO2 hy Cluster C, (3) Cluster C reduces Cluster B, and (4) Cluster B couples to external electron acceptors (133). 

Is the paramagnetic adduct between CO and Cluster A a kinetically intermediate in acetyl-CoA synthesis Questions have been raised about whether this adduct is a catalytic intermediate in the pathway of acetyl-CoA synthesis 187, 188) (as shown in Fig. 13), or is formed in a side reaction that is not on the main catalytic pathway for acetyl-CoA synthesis 189). A variety of biochemical studies have provided strong support for the intermediacy of the [Ni-X-Fe4S4l-CO species as the precursor of the carbonyl group of acetyl-CoA during acetyl-CoA synthesis 133, 183, 185, 190). These studies have included rapid ffeeze-quench EPR, stopped flow, rapid chemical quench, and isotope exchange. 

The subunits of CODH/ACS have been isolated (see earlier discussion). The isolated a subunit contains one Ni and four Fe and has spectroscopic properties (186) similar to those of Cluster A, the active site of acetyl-CoA synthesis (212). Unfortunately, it has no ACS activity. Therefore, ACS activity may reside in the a subunit or it may require both the a and the fi subunits. If Clusters B and/or C of the B subunit are involved in acetyl-CoA synthesis, one possible role could be in electron transfer. Although acetyl-CoA synthesis and the CO/ exchange reactions do not involve net electron transfer, both of these reactions are stimulated by ferredoxin, indicating that internal electron transfer within CODH/ACS may be required during the reaction (121). Further studies with the isolated subunits and the reconstitu-... 

The crystal structure of a CODH/ACS enzyme was reported only in 2002.43,44 It reveals a trio of Fe, Ni, and Cu at the active site (6). The Cu is linked to the Ni atom through two cysteine-S, the Ni being square planar with two terminal amide ligands. Planarity and amide coordination bear some resemblance to the Ni porphinoid in MCR. A two-metal ion mechanism is likely for acetyl CoA synthesis, in which a Ni-bound methyl group attacks an adjacent Cu—CO fragment with formation of a Cu-acyl intermediate. A methylnickel species in CODH/ACS has been identified by resonance Raman spectroscopy.45... 

Substrate availability for certain reactions can be optimized by anaplerotic ( topping-up ) reactions. For example, citrate synthase is a key control point of the TCA cycle. The co-substrates of citrate synthase are acetyl-CoA and oxaloacetate (OAA) and clearly, restriction in the availability of either substrate will decrease the rate of the citrate synthase reaction. Suppose, for example, a situation arises when acetyl-CoA concentration is significantly higher than that of OAA, the concentration of the latter can be topped-up and the concentration of acetyl-CoA simultaneously reduced by diverting some of the pyruvate away from acetyl-CoA synthesis (via pyruvate dehydrogenase) to OAA synthesis (via pyruvate carboxylase) as shown in Figure 3.1. The net effect is to balance the relative concentrations of the two co-substrates and thus to promote citrate synthase activity. 

With reference to divided attention, an association has been reported between blood glucose levels and performance on effortful dichotic listening paradigm (Parker Benton, 1995). While similar effects have been attributed to acetylcholine precursor (acetyl CoA) synthesis linked to glucose metabolism (Wenk, 1989), it is also possible that simple provision of metabolic substrates may account for such findings (Kennedy Scholey, 2000). 

The reaction [Eq. (7)] requires a disulfide-reducing system such as dithiothreitol or disulfide reductase and a reducing agent such as NADPH or reduced ferredoxin. It is proposed [Eq. (5)] that carbon monoxide oxidoreductase binds CO as a one-carbon intermediate [C,], which can be either oxidized to C02 or condensed with the methyl group of a methylated corrinoid protein and CoA in the final step of acetyl-CoA synthesis. 

The negative effects of acetyl-CoA on pyruvate dehydrogenase activity are supplemented by ATP and NADH. These effects are in the correct direction to cause the rate of acetyl-CoA synthesis to vary with the need for electrons and for regeneration of NADH and ATP. 

Well-defined nickel-sulfur complexes that enable a stepwise combination of CO, alkyl, and thiol groups to give thioesters can be anticipated to yield deeper insight into the molecular mechanism of the acetyl-CoA synthesis (142, 149). The complex [Ni(C3Me2—S4)] afforded an example for such a thioester synthesis. In principle, it is even catalytic and Scheme 36 summarizes the individual steps (10). 

The chemistry of acetyl-CoA synthesis is thought to resemble the Monsanto process for acetate synthesis in that a metal center binds a methyl group and CO and the CO undergoes a carbonyl insertion into the methyl-metal bond. Elimination of the acetyl group is catalyzed by a strong nucleophile, iodide in the industrial process and CoA in the biochemical one. Currently, there are two views of the catalytic mechanism. 

Grahame, D. A., and Demoll, E., 1995, Substrate and accessory protein requirements and thermodynamics of acetyl-CoA synthesis and cleavage in Methanosarcina barkeri, Biochem. 34(14) 4617n4624. 

Kumar, M., Lu, W.-P., and Ragsdale, S. W., 1994, Binding of carbon disufide to the site of acetyl-CoA synthesis by the nickel-iron-sulfur protein, CO dehydrogenase, from Clostridium thermoaceticum, Biochem. 33 976999777. 

Maynard, E. L., and Lindahl, P. A., 1999, Kinetic mechanism of acetyl-CoA synthesis catalyzed by CO dehydrogenase/acetyl-CoA synthase preliminary evidence for a molecular tutmel, J. Biol. Inorg. Chem. 74 227. 

Menon, S., and Ragsdale, S. W., 1998, Role of the [4Ee64S] cluster in reductive activation of the cobalt center of the corrinoid iron-sulfur protein from Clostridium thermoaceticum during acetyl-CoA synthesis Biochem. 37(16) 5689n5698. 

Ragsdale, S. W., and Riordan, C. G., 1996, The Role Of Nickel In Acetyl-CoA Synthesis By The Bifunctional Enzyme CO Dehydrogenase/Acetyl-CoA Synthase Enzymology And Model Chemistry, J. Bioinorganic Chemistry 1 4899493. 

Scheme 8 A proposed mechanism of acetyl CoA synthesis. (Reprinted from Proc. Natl. Set U.S.A., 2003,100, 3689-3694. 2003 National Academy of Science, U.S.A)...

Svetlitchnaia T, Svetlitchnyi V, Meyer O, Dobbek H. Structural insights into methyltransfer reactions of a corrinoid iron-sulfur protein involved in acetyl-CoA synthesis. Proc. Natl. Acad. Sci. U.S.A. 2006 103 14331-14336. 

---