Catalytic coupling of the active sites in acetyl-CoA synthase, a bifunctional CO-channeling enzyme.

Acetogenic bacteria contain acetyl-CoA synthase (ACS), an enzyme with two distinct nickel-iron-sulfur active sites connected by a tunnel through which CO migrates. One site reduces CO2 to CO, while the other synthesizes acetyl-CoA from CO, CoA, and the methyl group of another protein (CH3-CP). Rapid binding of CO2 and a two-electron reduction activates ACS. When CoA and CH3-CP bind ACS, CO is rerouted through the tunnel to the synthase site, and kinetic parameters at the reductase site are altered. Under these conditions, the rates of CO2 reduction and acetyl-CoA synthesis are synchronized by an ordered catalytic mechanism.     

Rapid kinetic studies of acetyl-CoA synthesis: evidence supporting the catalytic intermediacy of a paramagnetic NiFeC species in the autotrophic Wood-Ljungdahl pathway

CO dehydrogenase/acetyl-CoA synthase (CODH/ACS), a key enzyme in the Wood-Ljungdahl pathway of anaerobic CO(2) fixation, is a bifunctional enzyme containing CODH, which catalyzes the reversible two-electron oxidation of CO to CO(2), and ACS, which catalyzes acetyl-CoA synthesis from CoA, CO, and a methylated corrinoid iron-sulfur protein (CFeSP). ACS contains an active site nickel iron-sulfur cluster that forms a paramagnetic adduct with CO, called the nickel iron carbon (NiFeC) species, which we have hypothesized to be a key intermediate in acetyl-CoA synthesis. This hypothesis has been controversial. Here we report the results of steady-state kinetic experiments; stopped-flow and rapid freeze-quench transient kinetic studies; and kinetic simulations that directly test this hypothesis. Our results show that formation of the NiFeC intermediate occurs at approximately the same rate as, and its decay occurs 6-fold faster than, the rate of acetyl-CoA synthesis. Kinetic simulations of the steady-state and transient kinetic results accommodate the NiFeC species in the mechanism and define the rate constants for the elementary steps in acetyl-CoA synthesis. The combined results strongly support the kinetic competence of the NiFeC species in the Wood-Ljungdahl pathway. The results also imply that the methylation of ACS occurs by attack of the Ni(1+) site in the NiFeC intermediate on the methyl group of the methylated CFeSP. Our results indicate that CO inhibits acetyl-CoA synthesis by inhibiting this methyl transfer reaction. Under noninhibitory CO concentrations (below 100 microM), formation of the NiFeC species is rate-limiting, while at higher inhibitory CO concentrations, methyl transfer to ACS becomes rate-limiting.     

Evidence for intersubunit communication during acetyl-CoA cleavage by the multienzyme CO dehydrogenase/acetyl-CoA synthase complex from Methanosarcina thermophila. Evidence that the beta subunit catalyzes C-C and C-S bond cleavage

The carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from Methanosarcina thermophila is part of a five-subunit complex consisting of alpha, beta, gamma, delta, and epsilon subunits. The multienzyme complex catalyzes the reversible oxidation of CO to CO(2), transfer of the methyl group of acetyl-CoA to tetrahydromethanopterin (H(4)MPT), and acetyl-CoA synthesis from CO, CoA, and methyl-H(4)MPT. The alpha and epsilon subunits are required for CO oxidation. The gamma and delta subunits constitute a corrinoid iron-sulfur protein that is involved in the transmethylation reaction. This work focuses on the beta subunit. The isolated beta subunit contains significant amounts of nickel. When proteases truncate the beta subunit, causing the CODH/ACS complex to dissociate, the amount of intact beta subunit correlates directly with the EPR signal intensity of Cluster A and the activity of the CO/acetyl-CoA exchange reaction. Our results strongly indicate that the beta subunit harbors Cluster A, a NiFeS cluster, that is the active site of acetyl-CoA cleavage and assembly. Although the beta subunit is necessary, it is not sufficient for acetyl-CoA synthesis; interactions between the CODH and the ACS subunits are required for cleavage or synthesis of the C-C bond of acetyl-CoA. We propose that these interactions include intramolecular electron transfer reactions between the CODH and ACS subunits.     

Acetate biosynthesis by acetogenic bacteria. Evidence that carbon monoxide dehydrogenase is the condensing enzyme that catalyzes the final steps of the synthesis

The purified carbon monoxide dehydrogenase from Clostridium thermoaceticum is the only protein required to catalyze an exchange reaction between carbon monoxide and the carbonyl group of acetyl-CoA. This exchange requires that the CO dehydrogenase bind the methyl, the carbonyl, and the CoA groups of acetyl-CoA, then equilibrate the carbonyl with CO in the solution and re-form acetyl-CoA. CoA is not necessary for the exchange and, in fact, inhibits the reaction. These studies support the view that CO dehydrogenase is the condensing enzyme that forms acetyl-CoA from its component parts. Carbon dioxide also exchanges with the C-1 of acetyl-CoA, but at a much lower rate than does CO. At 50 degrees C and pH 5.3, the optimal pH, the turnover number is 70 mol of CO exchanged per min/mol of enzyme. Low potential electron carriers are stimulatory. The Km app for stimulation by ferredoxin is 50-fold less than the value for flavodoxin. Neither ATP or Pi stimulate the exchange. The EPR spectrum of the CO-reacted enzyme is markedly changed by binding of CoA or acetyl-CoA. Arginine residues of the CO dehydrogenase appear to be involved in the active site, possibly by binding acetyl-CoA. Mersalyl acid, methyl iodide, 5,5-dithiobis-(2-nitrobenzoate), and sodium dithionite inhibit the exchange reaction. A scheme is presented to account for the role of CO dehydrogenase in the exchange reaction and in the synthesis of acetate.     

Autocatalytic activation of acetyl-CoA synthase

Acetyl-CoA synthase (ACS ACS/CODH CODH/ACS) from Moorella thermoacetica catalyzes the synthesis of acetyl-CoA from CO, CoA, and a methyl group of a corrinoid-iron-sulfur protein (CoFeSP). A time lag prior to the onset of acetyl-CoA production, varying from 4 to 20 min, was observed in assay solutions lacking the low-potential electron-transfer agent methyl viologen (MV). No lag was observed when MV was included in the assay. The length of the lag depended on the concentrations of CO and ACS, with shorter lags found for higher [ACS] and sub-saturating [CO]. Lag length also depended on CoFeSP. Rate profiles of acetyl-CoA synthesis, including the lag phase, were numerically simulated assuming an autocatalytic mechanism. A similar reaction profile was monitored by UV-vis spectrophotometry, allowing the redox status of the CoFeSP to be evaluated during this process. At early stages in the lag phase, Co²⁺FeSP reduced to Co⁺FeSP, and this was rapidly methylated to afford CH₃-Co³⁺FeSP. During steady-state synthesis of acetyl-CoA, CoFeSP was predominately in the CH₃-Co³⁺FeSP state. As the synthesis rate declined and eventually ceased, the Co⁺FeSP state predominated. Three activation reductive reactions may be involved, including reduction of the A- and C-clusters within ACS and the reduction of the cobamide of CoFeSP. The B-, C-, and D-clusters in the subunit appear to be electronically isolated from the A-cluster in the connected subunit, consistent with the ~70 Å distance separating these clusters, suggesting the need for an in vivo reductant that activates ACS and/or CoFeSP.Abbreviations ACS acetyl-CoA synthase, also known as CODH (carbon monoxide dehydrogenase) or CODH/ACS or ACS/CODH - CH₃-Co³⁺FeSP, Co²⁺FeSP, and Co⁺FeSP corrinoid-iron-sulfur protein with the cobalamin in the methylated 3+, unmethylated 2+, and unmethylated 1+ states - CoA coenzyme A - DTT dithiothreitol - H-THF or THF tetrahydrofolic acid or tetrahydrofolate - MT methyl transferase - MV methyl viologen     

Reductive activation of the coenzyme A/acetyl-CoA isotopic exchange reaction catalyzed by carbon monoxide dehydrogenase from Clostridium thermoaceticum and its inhibition by nitrous oxide and carbon monoxide

The final steps in the synthesis of acetyl-CoA by CO dehydrogenase (CODH) have been studied by following the exchange reaction between CoA and the CoA moiety of acetyl-CoA. This reaction had been studied earlier (Pezacka, E., and Wood, H. G. (1986) J. Biol. Chem. 261, 1609-1615 and Ramer, W. E., Raybuck, S. A., Orme-Johnson, W. H., and Walsh, C. T. (1989) Biochemistry 28, 4675-4680). The CoA/acetyl-CoA exchange activity was determined at various controlled redox potentials and was found to be activated by a one-electron reduction with half-maximum activity occurring at -486 mV. There is approximately 2000-fold stimulation of the exchange by performing the reaction at -575 mV relative to the rate at -80 mV. Binding of CoA to CODH is not sensitive to the redox potential; therefore, the reductive activation affects some step other than association/dissociation of CoA. We propose that a metal center on CODH with a midpoint reduction potential of less than or equal to -486 mV is activated by a one-electron reduction to cleave the carbonyl-sulfur bond and/or bind the acetyl group of acetyl-CoA. Based on a comparison of the redox dependence of this reaction with that for methylation of CODH (Lu, W-P., Harder, S. R., and Ragsdale, S. W. (1990) J. Biol. Chem. 265, 3124-3133) and CO2 reduction and formation of the Ni-Fe-C EPR signal (Lindahl, P. A., Münck, E., and Ragsdale, S. W. (1990) J. Biol. Chem. 265, 3873-3879), we propose that the assembly of the acetyl group of acetyl-CoA, i.e. binding the methyl group of the methylated corrinoid/iron-sulfur protein, binding CO, and methyl migration to form the acetyl-CODH intermediate, occur at the novel Ni-Fe3-4-containing site in CODH. CO has two effects on the CoA/acetyl-CoA exchange: it activates the reaction due to its reductive capacity and its acts as a noncompetitive inhibitor. We also discovered that the CoA/acetyl-CoA exchange was inhibited by nitrous oxide via an oxidative mechanism. In the presence of a low-potential electron donor, CODH becomes a nitrous oxide reductase which catalytically converts N2O to N2. This study combined with earlier results (Lu, W-P., Harder, S. R., and Ragsdale, S. W. (1990) J. Biol. Chem. 265, 3124-3133) establishes that the two-subunit form of CODH is completely active in all reactions known to be catalyzed by CODH.     

Controlled potential enzymology of methyl transfer reactions involved in acetyl-CoA synthesis by CO dehydrogenase and the corrinoid/iron-sulfur protein from Clostridium thermoaceticum

Many anaerobic bacteria fix CO2 via the Wood pathway of acetyl-CoA synthesis. Carbon monoxide dehydrogenase (CODH), also called acetyl-CoA synthase, accepts the methyl group from the methylated corrinoid/iron-sulfur protein (C/Fe-SP), binds a carbonyl group from CO, CO2, or the carboxyl of pyruvate, and binds coenzyme A. Then CODH catalyzes the synthesis of acetyl-CoA from these enzyme-bound groups. Here, we have characterized the methyl transfer steps involved in acetyl-CoA synthesis. We have studied the reactions leading to methylation of CODH by methyl iodide and shown an absolute requirement of the C/Fe-SP in this reaction. In addition, we have discovered and partly characterized two previously unknown exchange reactions catalyzed by CODH: between the methylated C/Fe-SP and methylated CODH and between methylated CODH and the methyl moiety of acetyl-CoA. We have performed these two exchange reactions, methylation of the C/Fe-SP, and methylation of CODH at controlled potentials. The rates of all these reactions except the exchange between methylated C/Fe-SP and methylated CODH are accelerated (from 1 to 2 orders of magnitude) when run at low potentials. Our results provide strong evidence for a nucleophilic redox-active metal center on CODH as the initial acceptor of the methyl group from the methylated C/Fe-SP. This metal center also is proposed to be involved in the cleavage of acetyl-CoA in the reverse reaction.     

The role of nickel in acetyl-CoA synthesis by the bifunctional enzyme CO dehydrogenase/acetyl-CoA synthase: enzymology and model chemistry

 CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) is one of the four known nickel enzymes. It is a bifunctional protein that catalyzes the oxidation of CO to CO₂ at a nickel iron-sulfur cluster (Cluster C) and a remarkable condensation reaction between a methyl group (donated from a methylated corrinoid iron-sulfur protein), carbon monoxide, and coenzyme A to form acetyl-CoA at a separate nickel iron-sulfur cluster (Cluster A). This review focuses on the current understanding of the structure and function of Cluster A and on related model chemistry. It describes studies that uncovered the first example of a biological organometallic reaction sequence. The mechanism of acetyl-CoA synthesis includes enzymebound methylnickel, iron-carbonyl, and acylmetal intermediates. Discovery of the methylnickel species constituted the first example of an alkylnickel species in biology and unveiled a new biological role for nickel. Received: 10 April 1996 / Accepted: 4 July 1996     

Characterization of the acyl substrate binding pocket of acetyl-CoA synthetase

AMP-forming acetyl-CoA synthetase [ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1] catalyzes the activation of acetate to acetyl-CoA in a two-step reaction. This enzyme is a member of the adenylate-forming enzyme superfamily that includes firefly luciferase, nonribosomal peptide synthetases, and acyl- and aryl-CoA synthetases/ligases. Although the structures of several superfamily members demonstrate that these enzymes have a similar fold and domain structure, the low sequence conservation and diversity of the substrates utilized have limited the utility of these structures in understanding substrate binding in more distantly related enzymes in this superfamily. The crystal structures of the Salmonella enterica ACS and Saccharomyces cerevisiae ACS1 have allowed a directed approach to investigating substrate binding and catalysis in ACS. In the S. enterica ACS structure, the propyl group of adenosine 5'-propylphosphate, which mimics the acyl-adenylate intermediate, lies in a hydrophobic pocket. Modeling of the Methanothermobacter thermautotrophicus Z245 ACS (MT-ACS1) on the S. cerevisiae ACS structure showed similar active site architecture, and alignment of the amino acid sequences of proven ACSs indicates that the four residues that compose the putative acetate binding pocket are well conserved. These four residues, Ile312, Thr313, Val388, and Trp416 of MT-ACS1, were targeted for alteration, and our results support that they do indeed form the acetate binding pocket and that alterations at these positions significantly alter the enzyme's affinity for acetate as well as the range of acyl substrates that can be utilized. In particular, Trp416 appears to be the primary determinant for acyl chain length that can be accommodated in the binding site.     

Acetyl coenzyme A binding by chloramphenicol acetyltransferase: long-range electrostatic determinants of coenzyme A recognition.

The possible involvement of arginyl and lysyl side chains of chloramphenicol acetyltransferase (CAT) in binding coenzyme A (CoA) was studied by means of chemical modification, site-directed mutagenesis, variation in ionic strength, use of competitive inhibitors or substrate analogues, and X-ray crystallography. Unlike a number of enzymes, including citrate synthase, CAT does not employ specific ion pairs with the phosphoanionic centers of CoA to bind the acetyl donor, and arginyl residues play no role in recognition of the coenzyme. Although phenylglyoxal inactivates CAT reversibly, it does so by the formation of an unstable adduct with a thiol group, that of Cys-31 in the chloramphenicol binding site. The inhibitory effect of increasing ionic strength on kcat/Km(acetyl-CoA) can be explained by long-range electrostatic interactions between CoA and the epsilon-amino groups of Lys-54 and Lys-177, both of which are solvent-accessible. The epsilon-amino group of Lys-54 contributes 1.3 kcal.mol-1 to the binding of acetyl-CoA via interactions with both the 3'- and 5'-phosphoanions of CoA. Lys-177 contributes only 0.4 kcal.mol-1 to the productive binding of acetyl-CoA, mediated by long-range (approximately 14 A) interactions with the 5'-alpha- and -beta-phosphoanions of CoA. The combined energetic contribution of Lys-54 and Lys-177 to acetyl-CoA binding (1.7 kcal.mol-1) is less than that previously demonstrated (2.4 kcal.mol-1) for a simple hydrophobic interaction between Tyr-178 and the adenine ring of CoA (Day & Shaw, 1992).(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of carbon monoxide dehydrogenase in acetate synthesis by the acetogenic bacterium, Acetobacterium woodii

Carbon monoxide dehydrogenase (CODH) plays a key role in acetate synthesis by the acetogenic bacterium, Clostridium thermoaceticum. Acetobacterium woodii, like C. thermoaceticum contains high levels of CODH. In this work we show that crude extracts of A. woodii synthesize acetate from methyl tetrahydrofolate or methyl iodide, carbon monoxide and coenzyme A (CoA). The purified CODH from A. woodii catalyzes an exchange reaction between CO and the carbonyl group of acetyl-CoA even faster than the C. thermoaceticum enzyme, indicating the CODH of A. woodii, like that of C. thermoaceticum is an acetyl-CoA synthetase. Fluorescence and EPR studies further support this postulate by demonstrating that CODH binds CoA near the CO binding site involving a tryptophan residue. The UV absorption spectra and the amino acid compositions of A. woodii and C. thermoaceticum CODHs are very similar. Evidence is presented using purified enzymes from A. woodii that the synthesis of acetyl-CoA occurs by a pathway similar to that utilized by C. thermoaceticum.     

Kinetic characterization of the carbon monoxide-acetyl-CoA (carbonyl group) exchange activity of the acetyl-CoA synthesizing CO dehydrogenase from Clostridium thermoaceticum

CO dehydrogenase from Clostridium thermoaceticum is a nickel-containing enzyme that catalyzes both the reversible conversion of CO2 to CO (for incorporation into the carbonyl group of acetate) and the synthesis of acetyl-CoA from methyl corrinoid, CO, and CoASH. The latter activity is conveniently assayed by monitoring the exchange of [1-14C]acetyl-CoA (carbonyl group) with 12CO. Kinetic parameters for the highly oxygen sensitive exchange activity have been determined: Km (acetyl-CoA) = 600 microM; Vmax = 440 min-1. In addition, coenzyme A analogues have been tested as inhibitors of the exchange to probe the active site of the enzyme; each has no effect on the CO2 in equilibrium CO activity of CO dehydrogenase. Coenzyme A, the substrate for acetate biosynthesis, is a potent competitive inhibitor, KI = 7 microM. Comparison of this value with that for desulfo-CoA (KI = 6000 microM) suggests that a key mode of binding is through the sulfur atom, possibly to a metal site on the enzyme. The relatively high affinity of the enzyme for CoASH relative to acetyl-CoA is consistent with its proposed operation in the acetogenic direction. The differential sensitivity to oxygen and storage of the two activities of CO dehydrogenase as well as the contrasting effect of coenzyme A inhibitors suggests that acetate assemblage occurs at a site distinct from that for CO dehydrogenation.     

Analysis of Streptomyces coelicolor phosphopantetheinyl transferase, AcpS, reveals the basis for relaxed substrate specificity

The transfer of the phosphopantetheine chain from coenzyme A (CoA) to the acyl carrier protein (ACP), a key protein in both fatty acid and polyketide synthesis, is catalyzed by ACP synthase (AcpS). Streptomyces coelicolor AcpS is a doubly promiscuous enzyme capable of activation of ACPs from both fatty acid and polyketide synthesis and catalyzes the transfer of modified CoA substrates. Five crystal structures have been determined, including those of ligand-free AcpS, complexes with CoA and acetyl-CoA, and two of the active site mutants, His110Ala and Asp111Ala. All five structures are trimeric and provide further insight into the mechanism of catalysis, revealing the first detailed structure of a group I active site with the essential magnesium in place. Modeling of ACP binding supported by mutational analysis suggests an explanation for the promiscuity in terms of both ACP partner and modified CoA substrates.     

Enzymology of the acetyl-CoA pathway of CO2 fixation

We know of three routes that organisms have evolved to synthesize complex organic molecules from CO2: the Calvin cycle, the reverse tricarboxylic acid cycle, and the reductive acetyl-CoA pathway. This review describes the enzymatic steps involved in the acetyl-CoA pathway, also called the Wood pathway, which is the major mechanism of CO2 fixation under anaerobic conditions. The acetyl-CoA pathway is also able to form acetyl-CoA from carbon monoxide. There are two parts to the acetyl-CoA pathway: (1) reduction of CO2 to methyltetrahydrofolate (methyl-H4folate) and (2) synthesis of acetyl-CoA from methyl-H4folate, a carboxyl donor such as CO or CO2, and CoA. This pathway is unique in that the major intermediates are enzyme-bound and are often organometallic complexes. Our current understanding of the pathway is based on radioactive and stable isotope tracer studies, purification of the component enzymes (some extremely oxygen sensitive), and identification of the enzyme-bound intermediates by chromatographic, spectroscopic, and electrochemical techniques. This review describes the remarkable series of enzymatic steps involved in acetyl-CoA formation by this pathway that is a key component of the global carbon cycle.     

Interaction of ferredoxin with carbon monoxide dehydrogenase from Clostridium thermoaceticum.

Acetogenic bacteria, as determined with Clostridium thermoaceticum, synthesize acetate by the acetyl-CoA pathway which involves the reduction of CO2 to a methyl group and then combination of the methyl with CoA and a carbonyl group formed from CO or CO2 (Wood, H.G., Ragsdale, S.W., and Pezacka, E. (1986) Trends Biochem. Sci. 11, 14-18). Carbon monoxide dehydrogenase (CODH), the key enzyme in this pathway not only catalyzes the oxidation of CO to CO2 but also the final step, the synthesis of acetyl-CoA from a methyl group, CO, and CoA. Previously, it has been shown that ferredoxin can stimulate exchange of CO with CH3 14COSCoA (Ragsdale, S.W., and Wood, H.G. (1985) J. Biol. Chem. 260, 3970-3977). In the present study, it has been observed that ferredoxin and CODH can form an electrostatically stabilized complex. In order to identify the ferredoxin binding region on CODH, the ferredoxin and CODH were cross-linked by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The cross-linked CODH-ferredoxin adduct was enzymatically as active as the uncross-linked complex. The native CODH and cross-linked CODH-ferredoxin complex were subjected to cyanogen bromide cleavage. By comparison of the high-performance liquid chromatography peptide profiles, it was observed that the mobility of at least one peptide is altered in the CODH-ferredoxin cross-linked complex. The peptide was identified with residues 229-239 of the alpha-subunit of CODH.     

Analysis of the mechanism of chloramphenicol acetyltransferase by steady-state kinetics. Evidence for a ternary-complex mechanism.

The mechanism of the enzymic reaction responsible for chloramphenicol resistance in bacteria was examined by steady-state kinetic methods. The forward reaction catalysed by chloramphenicol acetyltransferase leads to inactivation of the antibiotic. Use of alternative acyl donors and acceptors, as well as the natural substrates, has yielded data that favour the view that the reaction proceeds to the formation of a ternary complex by a rapid-equilibrium mechanism wherein the addition of substrates may be random but a preference for acetyl-CoA as the leading substrate can be detected. Chloramphenicol and acetyl-CoA bind independently, but the correlation between directly determined and kinetically derived dissociation constants is imperfect because of an unreliable slope term in the rate equation. The reverse reaction, yielding acetyl-CoA and chloramphenicol, was studied in a coupled assay involving citrate synthase and malate dehydrogenase, and is best described by a rapid-equilibrium mechanism with random addition of substrates. The directly determined dissociation constant for CoA is in agreement with that derived from kinetic measurements under the assumption of an independent-sites model.     

Synthesis of acetyl coenzyme A from carbon monoxide, methyltetrahydrofolate, and coenzyme A by enzymes from Clostridium thermoaceticum

Two purified fractions from Clostridium thermoaceticum are shown to catalyze the following reaction: CO + CH3THF + CoA ATP leads to CH3COCoA + THF. The methyltetrahydrofolate (CH3THF) gives rise to the methyl group of the acetyl-coenzyme A (CoA) and the carbon monoxide (CO) and CoA to its carboxyl thio ester group. The role of ATP is unknown. One of the protein fractions (F2) is a methyltransferase, whereas the other fraction (F3) contains CO dehydrogenase and a methyl acceptor which is postulated to be a corrinoid enzyme. The methyltransferase catalyzes the transfer of the methyl group to the methyl acceptor, and the CO is converted to a formyl derivative by the CO dehydrogenase. By a mechanism that is as yet unknown, the formyl derivative in combination with CoA and the methyl of the methyl acceptor are converted to acetyl-CoA. It is also shown that fraction F3 catalyzes the reversible exchange of 14C from [1-14C]acetyl-CoA into 14CO and that ATP is required, but not the methyltransferase. It is proposed that these reactions are part of the mechanism which enables certain autotrophic bacteria to grow on CO. It is postulated that CH3THF is synthesized from CO and tetrahydrofolate which then, as described above, is converted to acetyl-CoA. The acetyl-CoA then serves as a precursor in other anabolic reactions. A similar autotropic pathway may occur in bacteria which grow on carbon dioxide and hydrogen.     

Purification and properties of acetyl-CoA:L-glutamate N-acetyltransferase from human liver.

Acetyl-CoA:L-glutamate N-acetyltransferase (amino acid acetyltransferase, EC 2.3.1.1) was isolated from human liver mitochondria by precipitation with (NH4)2SO4 and chromatography on hydroxyapatite, DEAE-cellulose and Sephacryl 300. This gave a 360-fold purification. The molecular weight was estimated to be approx. 190 000. The kinetic properties in the absence of arginine are compatible with a rapid-equilibrium random Bi Bi mechanism. The estimated constants are: for the substrates Km,acetyl-CoA 4.4 mM, Ki,acetyl-CoA 4.7 mM, Km,glutamate 8.1 mM, Ki,glutamate 8.8 mM; for the products, Ki,acetylglutamate 0.28 mM, Ki,CoA 5.6 mM. The rate constant for the forward direction is 1.24s-1. If in vivo the constants are of the same order of magnitude as in vitro, the synthesis of N-acetylglutamate, an obligate activator of the first step of urea synthesis, can be expected to occur in the mitochondrion under conditions where the amino acid acetyltransferase is not saturated by its substrates. The regulation of the first step of urea synthesis could thus depend mainly on the intramitochondrial substrate and perhaps product concentrations of amino acid acetyltransferase.     

The acetyl-CoA pathway of autotrophic growth

Abstract The most direct conceivable route for synthesis of multicarbon compounds from CO₂ is to join two molecules of CO₂ together to make a 2-carbon compound and then polymerize the 2-carbon compound or add CO₂ successively to the 2-carbon compound to make multicarbon compounds. Recently, it has been demonstrated that the bacterium, Clostridium thermoaceticum , grows autotrophically by such a process. The mechanism involves the reduction of one molecule of CO₂ to a methyl group and then its combination with a second molecule of CO₂ and CoA to form acetyl-CoA. We have designated this autotrophic pathway the acetyl-CoA pathway [1]. Evidence is accumulating that this pathway is utilized by other bacteria that grow with CO₂ and H₂ as the source of carbon and energy. This group includes bacteria which, like C. thermoaceticum , produce acetate as a major end product and are called acetogens or acetogenic bacteria. It also includes the methane-producing bacteria and sulfate-reducing bacteria.
The purpose of this review is to examine critically the evidence that the acetyl-CoA pathway occurs in other bacteria by a mechanism that is the same or similar to that found in C. thermoaceticum . For this purpose, the mechanism of the acetyl-CoA pathway, as found in C. thermoaceticum , is described and hypothetical mechanisms for other organisms are presented based on the acetyl-CoA pathway of C. thermoaceticum . The available data have been reviewed to determine if the hypothetical schemes are in accord with presently known facts. We conclude that the formation of acetyl-CoA by other acetogens, the methanogens and sulphate-reducing bacteria occurs by a mechanism very similar to that of C. thermoaceticum .     

Life with CO or CO2 and H2 as a source of carbon and energy

An account is presented of the recent discovery of a pathway of growth by bacteria in which CO or CO2 and H2 are sources of carbon and energy. The Calvin cycle and subsequently other cycles were discovered in the 1950s, and in each the initial reaction of CO2 involved adding CO2 to an organic compound formed during the cyclic pathway (for example, CO2 and ribulose diphosphate). Studies were initiated in the 1950s with the thermophylic anaerobic organism Clostridium thermoaceticum, which Barker and Kamen had found fixed CO2 in both carbons of acetate during fermentation of glucose. The pathway of acetyl-CoA biosynthesis differs from all others in that two CO2 are combined with coenzyme A (CoASH) forming acetyl CoA, which then serves as the source of carbon for growth. This mechanism is designated the acetyl CoA pathway and some have called it the Wood pathway. A unique feature is the role of the enzyme carbon monoxide dehydrogenase (CODH), which catalyzes the conversion of CoASH, CO, and a methyl group to acetyl CoA, the final step of the pathway. The pathway involves the reduction of CO2 to formate, which then combines with tetrahydrofolate (THF) to form formyl THF. It in turn is reduced to CH3-THF. The methyl is then transferred to the cobalt on a corrinoid-containing enzyme. From there the methyl is transferred to CODH, and CO and CoASH bind with the enzyme at separate sites. Acetyl CoA is then synthesized. CODH would more properly be called carbon monoxide dehydrogenase-acetyl CoA synthase as it catalyzes oxidation of CO to CO2 and the synthesis of acetyl CoA. The solution of the mechanism of this pathway required more than 30 years, in part because the intermediate compounds are bound to enzymes, the enzymes are extremely sensitive to O2 and must be isolated under strictly anerobic conditions, and the role of a corrinoid and CODH was unprecedented. It is now apparent that this pathway occurs (perhaps with some modification) in many bacteria including the methane and sulfur bacteria. In some humans this pathway is catalyzed by the bacteria of the gut and acetate is produced rather than methane; it is calculated that 2.3 x 10(6) metric tons of acetate are formed daily from CO2. A similar synthesis occurs in the hind gut of termites. It is becoming apparent that the acetyl CoA pathway plays a significant role in the carbon cycle.(ABSTRACT TRUNCATED AT 400 WORDS)