The LipidWeb blank

Coenzyme A, Acyl Carrier Protein and
Functionally Related Molecules

Before a fatty acid can be metabolized in tissues, for example by being esterified, oxidized or subjected to synthetic modification, it must usually be activated by conversion to a coenzyme A ester or acyl-CoA, with the fatty acid group linked to the terminal thiol moiety of CoA. This is true for the most primitive organisms, such as Archaea, through to humans (with the exception of certain bacteria discussed below). A thiol ester is a highly energetic bond that permits a facile transfer of the acyl group to receptor molecules, whether it is the simplest fatty acid of all, acetic acid, i.e. as acetyl-CoA, or one of the long-chain fatty acids, although alternative activating agents are known in some bacteria. Coenzyme A must be discussed in relation to the acyl carrier protein, which has the same functional moiety.

1.  Coenzyme A and Esters

Coenzyme A (CoASH or CoA) itself is a complex and highly polar molecule, consisting of adenosine 3',5'‑diphosphate linked to 4‑phosphopantetheinic acid (vitamin B5) and thence to β‑mercaptoethylamine, which is directly involved in acyl transfer reactions. The adenosine 3’,5’‑diphosphate moiety functions as a recognition site, increasing the affinity of CoA binding to enzymes. In mitochondria and peroxisomes, the concentrations of CoA are reported to lie in the range 2-5 mM and 0.7 mM, respectively, while that in the cytosol is much lower (0.05 to 0.14 mM). Although acyl-dephospho-CoA esters lacking the 3’‑phosphate group on the ribose moiety have been detected in tissues, their function is unknown.

Structural formula of Coenzyme A

The genes encoding the enzymes for CoA biosynthesis have been identified and the structures of many proteins in the pathway have been determined. Although there are sequence differences between prokaryotes and eukaryotes, coenzyme A is assembled in five steps from pantothenic acid, cysteine and ATP in essentially the same way in both groups. However, pantothenic acid per se can only be synthesised by microorganisms (including gut microflora) and plants, and animals must acquired it largely from the diet. In animals, CoA biosynthesis is believed to occur entirely in the cytosol of cells, and the first and rate-limiting step involves the enzyme pantothenate kinase, several isoforms of which are known.

Acyl-CoA synthesis and function: Intracellular free fatty acids arising from synthesis de novo or from the diet must be activated by thioesterification by a fatty acyl-CoA synthetase (fatty acid:CoA ligase) before they can be utilized for the synthesis of triacylglycerols, wax esters, long-chain aldehydes and alcohols, and complex lipids, or for covalent modification of proteins by myristoylation or palmitoylation by reactions involving many different N-acyltransferases. Indeed, CoA is intimately associated with most reactions of fatty acids, i.e. those involving elongases and desaturases, dehydrogenases, acyl-CoA thioesterases, carnitine-palmitoyltransferases, and lipid and protein acyltransferases. These functions are discussed in greater detail in most of the web pages in the Lipid Essentials section of this website. As these competing pathways are often present within a single subcellular compartment, there must be a high level of organization of the various enzymes that extends beyond their functions.

Acyl CoA synthetases activate fatty acids through a process that is energy-dependent and requires ATP and CoA. It is a two-stage reaction, requiring magnesium ions in the first step, which involves the formation of an acyl-AMP intermediate to react with CoA. In the process, ATP is consumed and AMP and pyrophosphate are produced.

Biosynthesis of coenzyme A esters

Acyl-CoA synthetases have been identified in humans with specificities for fatty acids in different chain-length groups, i.e. short-chain (2 to 3 carbons), medium-chain (4 to 12 carbons), long-chain (12 to 22 carbons), so-called ‘bubble-gum’ (14 to 24 carbons) and very-long-chain (18 to 26 or more carbons) fatty acid substrates. They are membrane-bound enzymes and are distinguished by two highly conserved sequence elements, i.e. an ATP/AMP binding motif, which is common to enzymes that form an adenylated intermediate, and a fatty acid binding motif. In all forms of life, multiple isoforms of these enzymes are known to be present, and six have been identified in the yeast genome while there are at least 26 in the human genome.

In humans, five long-chain acyl-CoA synthetases (ACSL1, -3, -4, -5, and -6) (EC convert fatty acids of 12-20 carbons to their corresponding CoA esters, with each isoform functionally differentiated by its substrate preference, regulatory mechanism, binding partner, expression pattern across tissues, and subcellular location. These factors control the ability of an individual ACSL to channel fatty acids towards different metabolic fates. For example, ACSL1 is anchored to the endoplasmic reticulum or to the outer mitochondrial membrane by a single N-terminal transmembrane domain. In the liver, that in the outer mitochondrial membrane directs the products largely towards oxidation, while the same isoform in the endoplasmic reticulum interacts with a large number of cellular proteins, of which some are concerned with esterification, including acyl-CoA binding proteins and ceramide synthase isoforms, and some in peroxisomes with oxidation. ACSL3 appears to have a preference for palmitic acid. ACSL4 uses arachidonic acid preferentially as its substrate and is involved in the remodelling of those phospholipids that incorporate this fatty acid and so may be connected to inflammatory responses. By mopping up excess arachidonic acid produced by the action of phospholipase A2, it may reduce eicosanoid biosynthesis. ACSL5 is the major intestinal ACSL isoform and is important for the absorption of fatty acids from dietary triacylglycerols and their subsequent metabolism, while ACSL6 is essential for the specific enrichment of docosahexaenoic acid (DHA) in brain lipids and is vital for neurological health. The latter also supplies this fatty acid selectively in seminiferous tubules and is required for male fertility in the mouse.

There is appreciable sequence homology between the very-long-chain acyl-CoA synthetases and certain fatty acid transport proteins in animals, so it is possible that they participate in the transport of fatty acyl moieties across membranes. Inevitably the effects of these enzymes on CoA levels and compositions have a bearing on a number of disease states, including metabolic disease and cancer.

Scottish thistleMany bacterial species, both Gram-negative and Gram-positive, synthesise long-chain acyl-CoA esters for lipid synthesis, and this enables them to make efficient use of exogenous fatty acids. In Escherichia coli, there are two inducible acyl-CoA synthetases, and fatty acid transport and activation are directly coupled to transcriptional control of the genes for various metabolic pathways for fatty acids; in this way, exogenous fatty acids repress synthesis de novo, for example. Acyl-CoA molecules form a separate pool from endogenously synthesized acyl-ACP, and they can be used for phospholipid synthesis or broken down by beta-oxidation, but cannot be used for lipopolysaccharide synthesis. Some species, including E. coli, use both acyl-CoA esters and acyl-ACPs for synthesis of phosphatidic acid de novo, and many other bacterial species activate fatty acids in a very different way, i.e. as the fatty acyl phosphates or adenylates (see below). However, other bacterial species do not make use of CoA in this way but instead utilize newly synthesised acyl groups (and even those of exogenous origin) linked via the thiol bond to the acyl carrier protein (ACP), i.e. in the form that they are produced by the type II fatty acid synthase (see below).

CoA esters are required for a number of processes in addition to esterification, including chain elongation and desaturation (some reactions in plants are exceptions), and these processes are discussed in other web pages. During fasting or starvation, intracellular long-chain fatty acids mobilized from adipose tissue reserves are catabolized as fuel by the mitochondrial β-oxidation pathway, and they must be first be converted to the CoA esters prior to synthesis of carnitine derivatives for translocation into the mitochondrion. Medium-chain fatty acids can enter mitochondria without carnitine transport but they must be still activated before β-oxidation can occur. Similarly, peroxisomes in animal cells have a distinct fatty acid β-oxidation system with a separate set of enzymes, including as many as three acyl-CoA oxidases. The acyl-CoA oxidase 1 catalyses the β-oxidation of straight-chain acyl-CoAs, while acyl-CoA oxidase 2 is involved in the oxidation of the side-chain of bile-acid precursors, and acyl-CoA oxidase 3 catalyses the oxidation of methyl branched-chain CoA esters. Activation is needed also for α-oxidation in tissues.

CoA is a key molecule in the catabolism of carbohydrates in the cytosol via the citric acid cycle in which acetyl-CoA is a major end-product. ATP citrate lyase is the primary enzyme responsible for this synthesis, and it catalyses the ATP-dependent and CoA-dependent cleavage of citrate to yield acetyl CoA, oxaloacetate, ADP and orthophosphate.

Action of ATP citrate lyase

Acetyl-CoA derived in this way or from acetate via a CoA synthetase in the cytosol is of course the primary precursor for lipogenesis, especially fatty acid synthesis (see our web page on saturated fatty acids, for example) and cholesterogenesis in animals. In plants, this pathway is less important for fatty acid biosynthesis as acetyl-CoA cannot cross into plastids, but it is vital for the biosynthesis of innumerable other lipid molecules, including sphingolipids, waxes, sterols and other isoprenoids.

Short-chain acyl-CoAs including free CoA, acetyl-CoA and malonyl-CoA are well known regulators of metabolic flux, with the ratio of acetyl-CoA to free CoA tightly regulating glycolysis and fatty acid oxidation. As well as its role in fatty acid synthesis, malonyl-CoA reduces fatty acid oxidation by inhibiting the transport of acyl-CoA into mitochondria. That derived from the acetyl-CoA synthetase in mitochondria is destined for oxidation. In addition to their role in lipid biosynthesis and catabolism, CoA esters have been shown to regulate the activities of a variety of enzymes, including that of acetyl-CoA carboxylase, an essential enzyme in fatty acid synthesis. Many genes and enzymes are regulated by deacylation and acylation via various short-chain acyl-CoAs, such as acetyl- and succinyl-CoA. Long-chain acyl-CoA esters also bind to certain hormone receptors and have a signalling function. While many of the effects observed for free fatty acids in nuclear signalling may also be attributable to acyl-CoA esters, this would take us into another very substantial realm of biochemistry outwith the mainstream of lipid biochemistry and function.

Acyl-CoA binding proteins: As they have both polar and hydrophobic molecular components, CoA esters of long-chain fatty acids have strong detergent-like physical properties with critical micellar concentrations of 5 to 42 µM, depending on their chain length and degree of unsaturation, and they have the potential to be disruptive towards cells. The intracellular concentration of free CoA and of acyl-CoA esters is tightly controlled by feedback inhibition of the acyl-CoA synthetase and by various extracellular stimuli, including nutrients, hormones and cellular metabolites. It is buffered by specific acyl-CoA binding proteins in the cytoplasm, such as that designated ACBP or DB1. which is an approximately 10 kDa protein that binds long-chain CoA esters with high specificity and affinity and reduces their effective concentration by up to 104 fold. ACBP is an intracellular acyl-CoA transporter, and it is also required for fatty acid chain elongation and sphingolipid synthesis in eukaryotes. Mitochondrial acyl-CoA concentrations are 10 fold higher than in the cytoplasm.

Six such proteins have been characterized in the model plant Arabidopsis that vary in their subcellular distribution, tissue-specificity, stress-responsiveness and ligand selectivity. They have also been studied intensively in oil seed crops. Acyl-CoA binding proteins contribute to the transport of fatty acids as acyl-CoAs from the chloroplasts to the endoplasmic reticulum for synthesis of complex lipids, or from the cytosol to the peroxisomes for fatty acid β-oxidation in order to provide the acetyl-CoA necessary for seed germination and seedling development, for example. One class of ACBPs is specific for very-long-chain acyl-CoAs, which it transports from the endoplasmic reticulum to and across the plasma membrane for the biosynthesis of surface lipids such as wax and cutin. These proteins are not simply transporters but are also important regulators in the synthesis of various signalling lipids that include phosphatidic acid, sterols, oxylipins, and sphingolipids.

Catabolism: At high concentrations, acyl-CoA are non-specific inhibitors of innumerable enzyme systems, and they must be removed from cells in part as their acyl-carnitine derivatives. In addition, there is a family of acyl-CoA thioesterases (ACOTs) of two main types, which are located in most cellular compartments and catalyse the hydrolysis of acyl-CoAs to the free fatty acid and coenzyme A. Suggested functions for these enzymes include ligand supply for nuclear receptors, regulation and termination of fatty acid oxidation in mitochondria and peroxisomes, and the control of the supply of acetate and of coenzyme A.

Pathological conditions, including certain hereditary conditions, that lead to impaired metabolism and accumulation of CoA and acyl-CoA within cells, trigger a sequence of reactions, which give rise to chronic illnesses.

2.  Acyl Carrier Protein

The 4-phosphopantetheine moiety, linked via its phosphate group to the hydroxyl group of serine, is the active component in another important molecule in lipid metabolism, Acyl Carrier Protein (ACP). This is a small (8.8 kDa) but ubiquitous and highly conserved carrier of acyl groups from active site to active site in polyketide and fatty acid synthases (see the web page on synthesis of fatty acids). A defining feature of an ACP is its flexibility in terms of its structure, substrate and enzyme partners. Thus, in animals, the ACPs are tethered covalently to the type I mega-synthase by flexible linkers in the peptide chain, which permit the intermediates to remain in an energy-rich linkage with access to spatially distinct enzyme-active sites in a manner that resembles an assembly line. However, the final step in fatty acid synthesis in most types of organism other than prokaryotes is transfer of the fatty acyl group from ACP to CoA.

In contrast, in type II fatty acid synthases in prokaryotes and plants, ACP-intermediates are diffusible discrete proteins that must deliver intermediates to the independent catalytic partners of the synthase in a concerted manner. They can also be used for production of other important cellular constituents, such as the octanoate moiety of lipoic acid and other biosynthetic products of acyl transfer, including non-ribosomal peptide and depsipeptide biosynthesis. ACP is also essential for the biosynthesis of rhamnolipids. In these organisms, the ACPs protect their cargo of reactive intermediates in the cytosol by sequestration within a hydrophobic cleft, which protects the thioester linkage from premature hydrolysis and other side reactions.

Biosynthesis of ACP involves post-translational transfer of a 4'-phosphopantetheinyl group from CoA to a conserved serine on apo-ACP, catalysed by a 4'-phosphopantetheinyl transferase (PPTase), to form holo-ACP (with 3',5'-bisphosphoadenosine as a byproduct). Acyl-acyl carrier protein synthetases act upon ACPs instead of coenzyme A, installing fatty acids onto the 4'-phosphopantetheine arm of holo-ACP with hydrolysis of ATP; they are distinct from the acyl-CoA ligases.

Biosynthesis of acyl-ACP

3.  Alternatives to CoA Esters

Acyl-phosphates: It has become apparent that most bacteria, including such important human Gram-positive pathogens as Streptococcus pneumoniae, Bacillus subtilis and Staphylococcus aureus, lack the glycerophosphate acyl transferase enzymes that make use of CoA. Instead, a fatty acyl-phosphate is the reactive acyl donor and can be produced by two routes both from fatty acids synthesised de novo and those of exogenous origin. In the first mechanism, the acyl-phosphate is produced by reaction of acyl-ACP with phosphate catalysed by an acyl-ACP:phosphate acyltransferase designated PlsX, and the product then requires a specific acyl-transferase, designated PlsY, so that it can be utilized in the first step for the synthesis of phosphatidic acid by acylation of position sn-1 of glycerol-3-phosphate; acyl-ACP is the donor for esterification of position sn-2. In these species, PlsX is key regulatory enzyme that synchronizes fatty acid synthesis with that of phospholipids. Although acyl-phosphates are less stable than thio esters in vitro, this is obviously not a problem in vivo.

Biosynthesis of acyl phosphates

The second route to acyl-phosphates, prior to incorporation of the acyl moieties into membrane phospholipids, is the only one to operate in S. aureus. In this instance, a fatty acid kinase (FakA) acting in concert with a fatty acid binding protein (FakB) uses ATP to phosphorylate unesterified fatty acids. In fact there are two binding proteins, FakB1 and FakB2, the first of which uses saturated fatty acids that are synthesised endogenously, while the second uses exogenous unsaturated fatty acids, such as oleic acid, derived from the host organism. The fatty acid kinase may be involved in controlling the production of virulence factors in S. aureus.

Acyl-adenylates: In Mycobacterium tuberculosis, fatty acyl-adenylates are produced by a fatty acyl-AMP ligase, which is similar in sequence to fatty acyl-coenzyme A (CoA) ligases. However, while the latter perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors as described above, fatty acyl-AMP ligases produce only the acyl-adenylates and do not continue to the second step. Fatty acids activated as the acyl-adenylates are then transferred to the polyketide synthases that produce the mycolic acids and other unusual fatty acids of mycobacterial lipids.

4.  Analysis of Coenzyme A Esters

The profile of CoA esters in tissues can be an important indication of metabolic status, but because of their strongly amphipathic character, analysis is fraught with technical difficulties. The procedures cited below are typical of those in use (but we have no personal experience). Extraction from tissues presents problems, and it may even be necessary to add a specific binding protein to ensure quantitative recoveries. Having obtained an appropriate extract, methods are available to separate short- and longer-chain fractions, and individual components can then be resolved by reversed-phase high-performance liquid chromatography. However, quantification can be a further problem, as it is not a straightforward matter to produce true solutions of pure CoA esters as standards. Electrospray-ionization tandem mass spectrometry now appears to hold particular promise for analysis.

Recommended Reading

Lipid listings Credits/disclaimer Updated: September 13th, 2019 Author: William W. Christie LipidWeb icon