Interaction of acetyl-CoA fragments with rat liver acetyl-CoA carboxylase

The interaction of acetyl-CoA fragments with rat liver acetyl-CoA carboxylase has been studied. Dephosphorylated acetyl-CoA did not actually differ from acetyl-CoA in its substrate properties. Non-nucleotide analogues of the substrate, S-acetylpantatheine and it's 4'-phosphate, also possess substrate properties (Vmax = 1.5% and 15% of the maximal rate value of acetyl-CoA carboxylation, respectively). The nucleotide fragment in the acetyl-CoA molecule produces a marked effect on the thermodynamics of the substrate-enzyme interaction, and is apparently involved in activation and appropriate orientation of the acetyl group in the active site. The better substrate properties of S-acetylpantetheine 4'-phosphate and the inhibitory properties of pantetheine 4'-phosphate, compared to the unphosphorylated analogues, evidence an important role of the 5'-beta-phosphate of 3'-phosphorylated ADP residue in acetyl-CoA binding to the enzyme.     

Wheat acetyl-CoA carboxylase

The acetyl-CoA carboxylase present in both wheat germ and total wheat leaf protein contains ca. 220 kDa subunits. It is the major biotin-dependent carboxylase present in wheat chloroplasts. Active acetyl-CoA carboxylase purified from wheat germ is a homodimer with an apparent molecular mass of ca. 500 kDa. The enzyme from wheat germ or from wheat chloroplasts is sensitive to the herbicide haloxyfop at micromolar levels. The incorporation of ¹⁴C-acetate into fatty acids in freshly cut wheat seedling leaves provides a convenient in vivo assay for both acetyl-CoA carboxylase and haloxyfop.     

Multi-subunit acetyl-CoA carboxylases

Acetyl-CoA carboxylase (ACC) catalyses the first committed step of fatty acid synthesis, the carboxylation of acetyl-CoA to malonyl-CoA. Two physically distinct types of enzymes are found in nature. Bacterial and most plant chloroplasts contain a multi-subunit ACC (MS-ACC) enzyme that is readily dissociated into its component proteins. Mammals, fungi, and plant cytosols contain the second type of ACC, a single large multifunctional polypeptide. This review will focus on the structures, regulation, and enzymatic mechanisms of the bacterial and plant MS-ACCs.     

Rabbit liver acetyl-CoA synthetase.

Acetyl-CoA synthetase (EC 6.2.1.1) was assayed in subcellular fractions of rabbit liver homogenates. The activity was located almost exclusively in the cytosol. There was no decrease in activity when butyrate or propionate (each at 5--20 mM) were added to the assay medium.     

Molecular biology of acetyl-CoA metabolism

We have characterized the expression of potential acetyl-CoA-generating genes (acetyl-CoA synthetase, pyruvate decarboxylase, acetaldehyde dehydrogenase, plastidic pyruvate dehydrogenase complex and ATP-citrate lyase), and compared these with the expression of acetyl-CoA-metabolizing genes (heteromeric and homomeric acetyl-CoA carboxylase). These comparisons have led to the development of testable hypotheses as to how distinct pools of acetyl-CoA are generated and metabolized. These hypotheses are being tested by combined biochemical, genetic and molecular biological experiments, which is providing insights into how acetyl-CoA metabolism is regulated.     

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     

The polymerization of acetyl-CoA carboxylase

Citrate, an allosteric activator of acetyl-CoA carboxylase, induces polymerization of an inactive protomeric form of the enzyme into an active filamentous form composed of 10-20 protomers. The light-scattering properties of the carboxylase were used to study the kinetics of its polymerization and depolymerization. From stopped flow kinetic studies, we have established that polymerization is a second order process, with a second order rate constant of 597,000 M-1 s-1. There appear to be two steps which limit polymerization of the inactive carboxylase protomer: 1) a rapid citrate-induced conformational change which is independent of enzyme concentration and leads to an active protomeric form of the enzyme (Beaty, N. B., and Lane, M. D. (1983) J. Biol. Chem. 258, 13043-13050, preceding paper) and 2) the dimerization of the active protomer, which constitutes the first step of polymerization and is enzyme concentration-dependent. Dimerization is the rate-limiting step of acetyl-CoA carboxylase polymerization. Depolymerization of fully polymerized acetyl-CoA carboxylase is caused by malonyl-CoA, ATP X Mg, and Mg2+. Both malonyl-CoA and ATP X Mg (and HCO-3) compete with citrate in the maintenance of a given state of the protomer-polymer equilibrium apparently by carboxylating the enzyme to form enzyme-biotin-CO-2 which destablizes the polymeric form. Free citrate is the species responsible for polymerizing the enzyme and Mg2+ causes depolymerization of the enzyme by lowering the concentration of free citrate.     

Molecular biology of cytosolic acetyl-CoA generation

ATP citrate lyase (ACL) catalyses the ATP-dependent reaction between citrate and CoA to form oxaloacetate and acetyl-CoA. Our molecular characterizations of the cDNAs and genes coding for the Arabidopsis ACL indicate that the plant enzyme is heteromeric, consisting of two dissimilar subunits. The A subunit is homologous to the N-terminal third of the animal ACL, and the B subunit is homologous to C-terminal two-thirds of the animal ACL. Using both ACL-A- and ACL-B-specific antibodies and activity assays we have shown that ACL is located in the cytosol, and is not detectable in the plastids, mitochondria or peroxisomes. During seed development, ACL-A and ACL-B mRNA accumulation is co-ordinated with the accumulation of the cytosolic homomeric acetyl-CoA carboxylase mRNA. Antisense Arabidopsis plants reduced in ATP citrate lyase activity show a complex phenotype, with miniaturized organs, small cell size, aberrant plastid morphology and reduced cuticular wax. Our results indicate that ACL generates the cytosolic pool of acetyl-CoA, which is the substrate required for the biosynthesis of a variety of phytochemicals, including cuticular waxes and flavonoids.     

Phosphorylation of pea chloroplast acetyl-CoA carboxylase

We have examined whether chloroplast acetyl-CoA carboxylase is a phosphoprotein. Pea ( Pisum sativum ) chloroplasts were incubated in the presence of [γ- ³³ P]-ATP and radiolabeled proteins were examined after immunoprecipitation with antibodies against all four known subunits of heteromeric chloroplast acetyl-CoA carboxylase. The β-subunit of the carboxyltransferase was found to be labeled by ³³ P. Phosphoamino acid analysis of the immunoprecipitated β-subunit of the carboxyltransferase indicates that it is phosphorylated on serine residues. Incorporation of ³³ P into carboxyltransferase β-subunit decreased in chloroplasts transferred to dark conditions after labeling in the light. Dephosphorylation of pea chloroplast extracts by an alkaline phosphatase-agarose conjugate reduced in vitro acetyl-CoA carboxylase activity by 67%. Furthermore, while acetyl-CoA carboxylase activity and its carboxyltransferase half-reaction were reduced in dephosphorylated extracts, the biotin carboxylase half-reaction was not inhibited. The evidence presented here points to the carboxyltransferase β-subunit of chloroplast acetyl-CoA carboxylase as a candidate for regulation by protein phosphorylation/dephosphorylation.     

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 .     

Nonhomogeneous labeling of liver mitochondrial acetyl-CoA

The specific activity of carbons 1 and 2 of plasma acetoacetate has been used as a measure of the specific activity of liver mitochondrial acetyl-CoA in tracer studies. To test whether or not acetoacetate actually reflects acetyl-CoA, livers were perfused with a mixture of substrates that are converted to mitochondrial acetyl-CoA: 1 mM lactate, 0.2 mM pyruvate, 0.2 mM acetate, and, where indicated, 0.2 mM octanoate or 0.2 mM alpha-ketoisocaproate. In each experiment, one of these substrates was 13C-labeled. Labeling of mitochondrial acetyl-CoA was assessed by three methods: (i) molar percent enrichment of total tissue acetyl-CoA; (ii) molar percent enrichment of carbons 4 and 5 of tissue citrate, the precursor of which is acetyl-CoA; and (iii) molar percent enrichment of carbons 1 and 2 of perfusate ketone bodies. Nonhomogeneous labeling of liver mitochondrial acetyl-CoA occurred under most conditions, i.e. the enrichments of carbons 4 and 5 of citrate were different from enrichments of carbons 1 and 2 of ketone bodies. Thus, based upon our results obtained in perfused livers, we question the validity of measuring the labeling of carbons 1 and 2 of acetoacetate as a noninvasive probe of liver mitochondrial acetyl-CoA.     

Regulation of acetyl-CoA carboxylase by ADP-ribosylation

Because of certain similarities between acetyl-CoA carboxylase (ACC) and tubulin, and the recent demonstration of the ADP-ribosylation of tubulin by cholera toxin, we have investigated a potential role for ADP-ribosylation in the regulation of ACC activity. Incubation of purified rat liver ACC with cholera toxin in the presence of millimolar concentrations of [adenylate-32P]NAD results in a time-dependent incorporation of ADP-ribose into ACC of greater than 2 mol/mol of enzyme subunit, accompanied by a marked inactivation of enzyme activity. This effect is not mimicked by pertussis toxin, ADP-ribose, or ribose 5-phosphate. Incubation of labeled ACC with snake venom phosphodiesterase and alkaline hydrolysis release 32P-products tentatively identified by high-performance liquid chromatography as 5'-[32P]AMP and [32P]ADP-ribose, respectively. These data are consistent with a mono-ADP-ribosylation of ACC catalyzed by cholera toxin. Phosphodiesterase treatment of inactivated ACC partially restores enzyme activity. The effects of ADP-ribosylation of ACC are expressed both as a decrease in the enzyme Vmax and as an increase in the apparent Ka for citrate. These results suggest that ACC might be a substrate for endogenous ADP-ribosyltransferases and that this covalent modification could be an important regulatory mechanism for the modulation of fatty acid synthesis in vivo.     

Microbial acetyl-CoA metabolism and metabolic engineering

Recent concerns over the sustainability of petrochemical-based processes for production of desired chemicals have fueled research into alternative modes of production. Metabolic engineering of microbial cell factories such as Saccharomyces cerevisiae and Escherichia coli offers a sustainable and flexible alternative for the production of various molecules. Acetyl-CoA is a key molecule in microbial central carbon metabolism and is involved in a variety of cellular processes. In addition, it functions as a precursor for many molecules of biotechnological relevance. Therefore, much interest exists in engineering the metabolism around the acetyl-CoA pools in cells in order to increase product titers. Here we provide an overview of the acetyl-CoA metabolism in eukaryotic and prokaryotic microbes (with a focus on S. cerevisiae and E. coli), with an emphasis on reactions involved in the production and consumption of acetyl-CoA. In addition, we review various strategies that have been used to increase acetyl-CoA production in these microbes.     

Hepatic zonation of acetyl-CoA carboxylase activity.

The activities of several hepatic enzymes are preferentially zonated to the periportal or perivenous cells of the liver acinus. Employing dual-digitonin-pulse perfusion of rat liver in the study of acetyl-CoA carboxylase (ACC), we have identified a heretofore unrecognized feature of hepatic zonation, namely an intrahepatic gradient in enzyme specific activity. ACC activity shows a relative periportal localization in normally feeding rats, even when corrected for ACC protein mass. In contrast with results previously reported by us [Evans, Quistorff & Witters (1989) Biochem. J. 259, 821-829], the total mass of both hepatic ACC isoenzymes was not found to differ between the two hepatic zones in the present study. In perfusion eluates from fed animals, periportal ACC displays enhanced citrate reactivity and two kinetic components of acetyl-CoA reactivity; the largest periportal/perivenous gradient (5-fold) is accounted for by a species with a lower Km for acetyl-CoA. The zonal gradient in ACC maximal velocity, measured in eluates from fed rats, does not persist after ACC purification, although the isolated periportal enzyme, like dephosphorylated ACC, has a lower activation constant for citrate. Total ACC protein phosphatase activity is higher in periportal eluates, but no differences in the activities of either a 5'-AMP-activated ACC kinase or the cyclic-AMP-dependent protein kinase are noted between the hepatic zones. The induction of total hepatic ACC mass and specific activity, on fasting/refeeding with a high-carbohydrate diet, abolishes the periportal/perivenous activity gradient, largely owing to a selective activation of perivenous enzyme. Nutritional induction is also accompanied by a marked alteration in ACC acetyl-CoA kinetics and abolition of the gradient in total ACC phosphatase. These studies indicate that hepatic enzyme zonation, which is often attributed to differential expression of enzyme protein, may result from zonal variations in enzyme specific activity, owing to differences in allosteric regulation and/or covalent modification.     

Bimodal activation of acetyl-CoA carboxylase by glutamate

Acetyl-CoA carboxylase (ACC) catalyzes the formation of malonyl-CoA, an essential substrate for fatty acid biosynthesis and a potent inhibitor of fatty acid oxidation. Here, we provide evidence that glutamate may be a physiologically relevant activator of ACC. Glutamate induced the activation of both major isoforms of ACC, prepared from rat liver, heart, or white adipose tissue. In agreement with previous studies, a type 2A protein phosphatase contributed to the effects of glutamate on ACC. However, the protein phosphatase inhibitor microcystin LR did not abolish the effects of glutamate on ACC activity. Moreover, glutamate directly activated purified preparations of ACC when protein phosphatase activity was excluded. Phosphatase-independent ACC activation by glutamate was also reflected by polymerization of the enzyme as judged by size-exclusion chromatography. The sensitivity of ACC to direct activation by glutamate was diminished by treatment in vitro with AMP-activated protein kinase or cAMP-dependent protein kinase or by beta-adrenergic stimulation of intact adipose tissue. We conclude that glutamate, an abundant intracellular amino acid, induces ACC activation through complementary actions as a phosphatase activator and as a direct allosteric ligand for dephosphorylated ACC. This study supports the general hypothesis that amino acids fulfill important roles as signal molecules as well as intermediates in carbon and nitrogen metabolism.