CHEMICAL BIOLOGY

Chemistry of CoA-Dependent Enzymes

 

Dale G. Drueckhammer, Department of Chemistry, State University of New York at Stony Brook

doi: 10.1002/9780470048672.wecb093

 

Coenzyme A (CoA) and its thioesters play a diverse array of roles in biologic systems. The enzymes that catalyze reactions of CoA are of interest for a variety of reasons, including, but not limited to, their potential as drug targets. The enzymology of CoA biosynthesis is now well understood, and the genes for all of the enzymes involved have been identified. Thioesters are inherently reactive toward acyl transfer reactions and toward reactions involving deprotonation of the a-carbon, and these are the primary reactivity patterns in enzyme-catalyzed reactions of CoA thioesters. CoA utilizing enzymes have been widely studied mechanistically and structurally. Analogs of the natural CoA thioester substrates have been widely used in these studies, including thioesters of unnatural or uncommon acyl groups as well as a large number of analogs in which the thioester is replaced with alternative functionality. Recent technical applications of CoA have included the tagging of carrier protein domains and carrier protein fusions with tagged phosphopantetheine derivatives transferred enzymatically from the corresponding tagged CoA derivatives using promiscuous phosphopantetheinyl transferases.

 

Coenzyme A (CoA) is a cofactor that has been estimated to be used by about 4% of all enzymes, although more recent analysis of the BRENDA database (http://www.brenda.uni-koeln.de/) suggests the number may be closer to 9% (1). The biochemical pathways and processes involving CoA thioesters are diverse and widespread, whereas the kinds of reactions involved primarily follow the inherent reactivity of the thioester functionality. This article provides a brief overview of CoA biosynthesis and a summary of the common types of reactions of CoA thioesters. Also presented is a brief introduction to structural studies and a more extensive description of some types of analogs of natural CoA thioesters that have been employed as mechanistic probes for CoA using enzymes. The application of CoA derivatives and CoA biosynthetic enzymes for the tagging of carrier proteins and carrier protein fusions is also described.

 

Biologic Background

Coenzyme A (abbreviated CoA or CoASH, 6) was discovered by Lipmann in the 1940s, and its structure was first reported in 1953 (2, 3). The structure of CoA consists of 3'-phosphoadenosine and pantetheine, linked by a pyrophosphate group (Fig. 1). The pantetheine moiety is derived from pantothenic acid 1, also known as vitamin B5. CoA and its thioester derivatives are involved in a wide range of biologic processes, including the TCA or Krebs cycle, fatty acid biosynthesis and degradation, antibiotic resistance mechanisms, gene expression, hormone biosynthesis and regulation, and nerve impulse conductance. Numerous CoA utilizing enzymes are either established or potential drug targets. The enzymes HMG-CoA reductase and acyl-CoA cholesterol acyltransferase (ACAT), which are involved in cholesterol biosynthesis and metabolism, are targets of drugs for cholesterol management. Inhibitors of malonyl-CoA decarboxylase may act as cardioprotective agents (4), whereas fatty acid synthase inhibitors have been explored as antimicrobial agents (5). CoA thioesters may also be involved in fatty acid-induced insulin resistance (6). Acetyl-CoA carboxylase and other acyl-CoA carboxylases are potential targets for anticancer and antiobesity agents as well as are herbicidal targets (7). Thioesters of CoA serve as the building blocks for the polyketide synthases that make a wide variety of natural bioactive compounds and are also involved as building blocks and intermediates in the biosynthesis of natural bacterial polyesters (8, 9). The interesting organometallic mechanism of the nickel-containing enzyme carbon monoxide dehydrogenase, which forms acetyl-CoA from carbon monoxide, CoA, and a methyl group donor, has been the target of extensive fundamental mechanistic studies (10). CoA thioesters have also been studied in ribozyme-catalyzed reactions (11). Thus, the roles of CoA utilizing enzymes in biology and the reasons for their interest are widespread and diverse.

 

Chemistry and Reactivity of CoA

CoA is involved in the activation and transfer of acyl groups in a wide variety of enzymatic reactions. Whereas many coenzymes function as co-catalysts that remain bound to a single enzyme molecule, CoA acts as a diffusible carrier of acyl groups between different enzymes.

 

CoA biosynthesis

The biosynthesis of CoA from pantothenic acid is shown in Fig. 1. Extensive coverage of this topic and references to the original literature can be found in recent reviews (1, 12). Pantothenic acid 1 is phosphorylated by pantothenate kinase to form 2. At least three distinct types of pantothenate kinases from different organisms have been observed (1). The cysteamine moiety is introduced by initially coupling with L-cysteine catalyzed by the phosphopantothenylcysteine synthetase activity to form 3 followed by a decarboxylation reaction to form phosphopantetheine 4. These two steps are catalyzed by a single bifunctional enzyme in most prokaryotes but by distinct enzymes in higher organisms. The bacterial phosphopantothenylcysteine synthetase is CTP-dependent, whereas the plant and mammalian enzymes prefer ATP. 3 is not inherently prone to decarboxylation, but this reaction has recently been shown to occur by temporary oxidation of the thiol to the thioaldehyde, which readily undergoes decarboxylation to an enethiolate intermediate (13). Phosphopantetheine is coupled with ATP to form dephospho-coenzyme A 5 followed by phosphorylation of the 3'hydroxyl group to form CoA. The final two steps are catalyzed by a single bifunctional enzyme in mammals, although they are catalyzed by separate enzymes in bacteria (1). The genes coding for all of the enzymes of this pathway (coaA-E) have now been identified, as have the genes for the four enzymes of pantothenic acid biosynthesis in bacteria (1, 12, 14). Pantothenic acid biosynthesis has also been studied in plants (15). Pantothenate kinase is a key regulatory point in CoA biosynthesis and is subject to feedback inhibition by CoA in most species (1). Pantothenate kinase can also catalyze the phosphorylation of pantetheine and pantetheine analogs (16, 17), although phosphorylation occurs before coupling with L-cysteine in the biosynthetic pathway.

 

 

Figure 1. Biosynthesis of CoA from pantothenic acid. E1: pantothenate kinase (CoaA); E2: phosphopantothenoylcysteine synthetase (CoaB); E3: phosphopantothenoylcysteine decarboxylase (CoaC); E4: phosphopantetheine adenylyltransferase (CoaD); E5: dephosphocoenzyme A kinase (CoaE).

 

Reactions and processes involving CoA

The reactions of CoA involve only the thiol group and the acyl moieties attached to the thiol group as thioesters, with the remainder of the structure serving as a recognition element that facilitates binding to the appropriate enzymes. The notable exception is the phosphopanetheinyl transferases that catalyze transfer of the phosphopantetheine moiety of CoA to a serine hydroxyl group in the acyl carrier proteins involved in fatty acid and polyketide synthetases and the related peptidyl carrier proteins of the nonribosomal peptide synthetases (8, 18).

 

Inherent teactivity of thiols and thioesters

Thiols are structurally similar to alcohols and can generally undergo the same kinds of reactions. However, a typical thiol group (pKa 9-10) is substantially more acidic than the corresponding hydroxyl group (pKa 16). The thiolate ion is also much more nucleophilic than the corresponding alkoxide. This combination of acidity to form the thiolate and nucleophilicity of the thiolate facilitates both alkylation and acylation reactions of thiols, with acylation being the primarily relevant reaction of CoA. A thioester has two inherent modes of reactivity that are both observed in enzyme-catalyzed reactions of CoA thioesters. One common type of reaction is initiated by reaction of a basic group to remove a proton from the carbon alpha to the carbonyl to form an enolate nucleophile, which then reacts with an electrophilic substrate. The other common type of enzymatic reaction of CoA thioesters involves acyl transfer by attack of an acyl acceptor nucleophile at the thioester carbonyl carbon. The inherent characteristics of the thioester make it more reactive than esters or amides in both of these types of reactions. The a-protons of a typical thioester (pKa 21) are about 104-fold more acidic than those of an ester (pKa 25), whereas the a-protons of an amide are even less acidic than those of an ester (19). The free energy of hydrolysis of a thioester is about 2 kcal/mol greater than that of an ester and, again, the difference relative to an amide is even greater (20), which provides a thermodynamic driving force in acyl transfer from CoA to an alcohol or amine nucleophile, whereas acyl transfer reactions to carbanion nucleophiles are also common. Thioesters are also kinetically much more reactive than esters toward most nucleophiles including thiolates, amines, and carbanions, with amides being much less reactive even than esters (21, 22). However, the rates of reaction of esters and thioesters toward hydrolysis by aqueous base are essentially identical, with thioesters even appearing to be slightly less reactive than oxoesters in some examples (21). This unique reactivity of thioesters provides for their reasonable stability in aqueous solution despite their substantial thermodynamic reactivity and inherently high kinetic reactivity toward most nucleophiles.

 

Acyl-CoA synthetases and related enzymes

Acetyl-CoA is formed from CoA and acetate by the enzyme acetyl-CoA synthetase, an ADP-forming ligase. Phosphotrans- acetylase forms acetyl-CoA from CoA and acetyl-phosphate, which in turn is formed from acetate and ATP catalyzed by acetate kinase. Other enzymes that can form acetyl-CoA from CoA and other acetyl group donors include ATP citrate lyase and thiolase. Longer chain acyl-CoA thioesters are typically formed from CoA and a fatty acid catalyzed by ligases generally known as acyl-CoA synthetases.

 

Acyltransferases

Most acyltransferases catalyze acyl transfer to a hydroxyl or amine group of the acceptor substrate, whereas CoA ester hydrolysis by thioesterases is in effect acyl transfer to water.

Choline acetyltransferase catalyzes transfer of the acetyl group from acetyl-CoA to the hydroxyl group of choline. The product acetylcholine is a major neurotransmitter (23). Serotonin acetyltransferase controls the sleep cycle by catalyzing acetyl transfer to the primary amine group of serotonin, which is the rate-limiting step in melatonin biosynthesis (24). Chloramphenicol acetyltransferase catalyzes acetyl transfer to a primary hydroxyl group, which destroys the antibiotic activity of chloramphenicol thereby conferring antibiotic resistance, whereas the antibiotics gentamicin and kanamycin are similarly inactivated by acetytransferases (25). Other extensively studied acetyltransferases include the histone acetyltransferases (HATs), which mediate a major control element in gene expression (26). Acyltransferases are also known that catalyze acyl transfer from longer chain acyl-CoA. The highly studied protein N-myristoyltransferase catalyzes transfer of the myristoyl group to the amine group of N-terminal glycine of a protein substrate (27). The palmitoyltransferases catalyze transfer of palmitic acid to a cysteine thiol group of certain proteins (28). These posttranslational acyltransfer reactions are important in membrane anchoring of proteins and may play a role in signaling events (29). Carnitine palmitoyltransferase catalyzes acyl transfer to the hydroxyl group of carnitine (30). The two general mechanisms for acyltransferases are a direct transfer to the nucleophilic acceptor or a two-step process involving initial acyl transfer to a nucleophilic group (usually a cysteine thiol) of the enzyme to form an acyl enzyme intermediate. Examples of both types of mechanisms are well documented, although the direct transfer mechanism appears to be more common.

 

Claisen enzymes

The enzymes that form a nucleophilic enolate intermediate by deprotonation of the methyl group of acetyl-CoA, which then reacts with a second electrophilic substrate, are generally referred to as Claisen enzymes in the literature, although not all catalyze true Claisen condensation reactions. Some common examples of Claisen enzymes and their electrophilic substrates are shown in Fig. 2. Thiolase catalyzes a true Claisen condensation reaction in which the electrophilic substrate is a second equivalent of acetyl-CoA, whereas in enzymes recognizing longer chain substrates, the electrophilic substrate can be a medium or long chain acyl-CoA (31). From the perspective of the electrophilic substrate, thiolase is also an acyltransferase in which the acyl group is transferred to nucleophilic carbon. Acetyl-CoA carboxylase catalyses transfer of the carboxylate group from carboxybiotin to the enolate (32). Both citrate synthase and HMG-CoA synthase catalyze reactions of the enolate with an electrophilic carbonyl group of a ketone substrate in what is formally an aldol reaction (33). In both of these examples, the enzyme also catalyzes a subsequent thioester hydrolysis reaction to release free CoA. Malate synthase catalyzes a very similar reaction (34). Although an enol intermediate for the Claisen enzymes and other mechanistically related enzymes has gained some consideration, an enolate intermediate stabilized by hydrogen bonding or in some cases possibly metal ion coordination to the carbonyl oxygen seems generally preferred (35). The low barrier or short-strong hydrogen bond proposal would favor the proton of the hydrogen bond being equally shared between the substrate oxygen and the hydrogen bond donor group (36).

 

 

Figure 2. Some common Claisen enzymes.

 

Enzymes catalyzing reactions of the acyl moiety

Certain CoA thioester using enzymes catalyze reactions at the P-carbon or other carbons of the acyl group more distant from the thioester functionality. The fatty acid β-oxidation cycle provides some examples (Fig. 3). Fatty acids 7 enter the cycle by initial conversion to the CoA ester 8, which is then oxidized to the α, β-unsaturated thioester 9 by a flavin-dependent enzyme. Addition of water to the double bond to form the P-hydroxy thioester 10 is catalyzed by the enzyme crotonase, which is the centerpiece of the crotonase superfamily of enzymes that catalyze related reactions (37), which is followed by oxidation of the alcohol to form the β-keto thioester 11. A retro-Claisen reaction catalyzed by thiolase forms acetyl-CoA 12 along with a new acyl-CoA 13 having a carbon chain two carbons shorter than in the initial or previous cycle.

 

 

Figure 3. The fatty acid p-oxidation cycle. E1: acylcoenzyme A synthetase; E2: acylcoenzyme A dehydrogenase; E3: enoylcoenzyme A hydratase (crotonase); E4: p-hydroxyacylcoenzyme A dehydrogenase; E5: thiolase.

 

Chemical Tools and Techniques

Structural studies of CoA utilizing enzymes

There is no single sequence motif common to all CoA utilizing enzymes, although homology has been observed across some fairly broad subsets. X-ray crystallography has been a powerful tool in the structural elucidation of CoA utilizing enzymes, and some structural studies have also been performed using NMR. Crystal structures for several of the enzymes of CoA biosynthesis have been solved while the solved structures of different CoA and CoA ester utilizing enzymes in the protein data bank numbers near 100, not including multiple structures of different complexes of the same protein and of the same protein from multiple sources. The conformation of CoA thioesters in solution has also been studied by NMR and comparisons made to the enzyme-bound conformation (38).

 

Applications of analogs of natural CoA thioesters

Synthetic analogs of natural CoA ester substrates have been widely used as mechanistic probes in studies of CoA ester utilizing enzymes (39). The most readily available are CoA thioesters of unnatural or uncommon acyl groups prepared by simple acylation of CoA. A number of haloacyl-CoA derivatives have been prepared, including fluoroacetyl-CoA (40). Fluoroacetyl-CoA is accepted as a substrate by citrate synthase to form fluorocitrate, which is an inhibitor of aconitase. This enzymatic processing to fluorocitrate and resulting inhibition of aconitase is responsible for the high toxicity of fluoroacetate. Bromoacetyl-CoA 14 was shown to inhibit carnitine acetyltransferase in the presence of carnitine 15 as shown in Fig. 4. Enzyme-catalyzed transfer of the bromoacetyl group to carnitine to form bromoacetyl carnitine 16 is followed by bromide displacement by the nucleophilic thiol group of CoA to form the bisubstrate adduct 17, which is the actual inhibitory species that binds tightly to the enzyme (41). Similar bisubstrate adducts have been employed as inhibitors of serotonin acetyltransferase (24). Other halo-acyl CoA thioesters as well as CoA thioesters having an epoxide in the acyl moiety have been employed as reactive electrophiles to trap nucleophilic residues in the active site of their target enzymes. CoA thioesters of unsaturated acids have also been widely used. Hexadienoyl-CoA, cinnamoyl-CoA, and other α, β-unsaturated acyl-CoAs having extended conjugation or having a heteroatom in place of the γ-carbon have been employed as spectroscopic probes of their enzyme complexes. The additional unsaturation or the heteroatom substituent serve to shift the UV absorbance to longer wavelength and, in some cases, also shifts the equilibrium of a reversible enzyme-catalyzed reaction toward the a,P-unsaturated substrate (42, 43). 3-Alkynoyl-CoA thioesters 18 were used as mechanism-based inhibitors of thiolase, with enzyme-catalyzed isomerization to the allenoyl-CoA 19 being followed by conjugate addition of an active site nucleophile to form the inactive enzyme adduct 20 (Fig. 4) (44). This work was preceded by studies of inactivation of the dehydratase component of fatty acid synthase by 3-alkynoyl thioesters (45). In later work, simplified 3-alkynoyl pantetheine thioesters were employed similarly to identify the active site base in thiolase (46). Similar compounds have also been shown to inactivate acyl-CoA dehydrogenases and enoyl reductase (47, 48). Several CoA thioesters of acids bearing cyclopropyl groups have been studied with special interest in those that undergo cyclopropyl ring opening of radical intermediates (49-51). Various other CoA thioesters bearing functionality, including photoaffinity labels (52, 53) and nitroxide spin labels (54) in the acyl moiety, have been prepared and employed as mechanistic tools. The dithioester analogs of acetyl-CoA 21 and of fluoroacetyl-CoA and octanoyl-CoA were prepared and shown to have interesting spectral properties, with a λmax of 306 nm and enhanced acidity of the α-protons (pKa = 12.5) (55). 21 underwent very rapid exchange of the α-protons with solvent catalyzed by citrate synthase but reacted very slowly in the overall reaction of citrate synthase. CoA dithioesters have also been employed in studies of thiolase, HMG-CoA lyase and HMG-CoA reductase. Structure 22 is illustrative of a number of keto thioether analogs in which a methylene group is inserted between the sulfur and carbonyl carbon of the thioester. Such analogs are prepared by alkylation of CoA with a halomethyl ketone. These analogs do not have a cleavable bond between the CoA and acyl moiety and are often good inhibitors of acyltransferase enzymes (56). CoA has also been derivatized in the adenine base for attachment to a solid support for use in affinity chromatography (57, 58).

 

 

 

Figure 4. Some unnatural CoA thioesters and related analogs. E1: carnitine acetyltransferase; E2: thiolase.

 

CoA thioester analogs have also been prepared that cannot be made by derivatization of natural CoA. Some early examples were made by nonenzymatic synthesis (59) generally following the original synthesis of CoA developed by Moffatt and Khorana (60). More recently analogs have been made enzymatically using the enzymes of CoA biosynthesis. One approach uses pantothenate kinase, phosphopantetheine adenylyltransferase, and dephospho-CoA kinase to convert synthetic pantetheine analogs (e.g., 23) to the corresponding CoA analogs, with phospho-enolpyruvate and pyruvate kinase included to regenerate the ATP consumed in the kinase steps as shown in Fig. 5 for the synthesis of an oxoester analog 26 of crotonyl-CoA (16). The three biosynthetic enzymes have been shown to accept substrate analogs having significant modification relative to the natural substrates, including analogs having long chain acyl groups. This synthetic method takes advantage of the ability of pantothenate kinase to phosphorylate pantetheine and its analogs and derivatives in addition to the natural substrate pantothenic acid. The other primary synthetic approach has used the final enzymes of CoA biosynthesis to make a CoA analog 27 in which the outermost amide bond is replaced with a thioester group (61). This analog serves as a general synthon for other CoA analogs 28 by reaction with a primary amine bearing the functionality of interest in place of the thiol group of CoA (Fig. 6).

 

 

Figure 5. Enzymatic synthesis of a CoA oxoester.

 

 

 

Figure 6. Aminolysis of a general CoA analog synthon for CoA analog synthesis.

 

The oxoester analog of crotonyl-CoA 26 shown in Fig. 5 was used as an alternative substrate for crotonase and exhibited about 300-fold decreased activity relative to the natural thioester (16). Some other representative analogs are shown in Fig. 6. Several dethia analogs in which the thioester sulfur atom is replaced with a methylene group have been prepared, as represented by the acetyl-CoA analog 29 (59, 61). These analogs are generally inhibitors of enzymes that catalyze reactions involving cleavage of the thioester such as acyltransferases, while serving as substrates for enzymes catalyzing reactions that do not involve cleavage of the thioester such as some of the Claisen enzymes. The carboxylate analog 30 is representative of compounds prepared as stable mimics of the enolate (or enol) intermediate of the Claisen enzymes (61, 62). 30 was shown to be a potent inhibitor of citrate synthase, binding about 1000-fold more strongly than acetyl-CoA, and the structure of the enzyme-inhibitor complex was solved (33). The analog 31 having the orientation of the thioester reversed was shown to be an inhibitor of thiolase, apparently forming an acyl-enzyme with the CoA moiety in a step mimicking the formation of the acetyl-enzyme intermediate from acetyl-CoA (63).

 

 

Figure 7. Protein tagging using a phosphopantetheinyl transferase (PPTase) along with CoA biosynthetic enzymes (CP = carrier protein).

 

Other Technical Applications of CoA

A recent interesting application of CoA and CoA analogs has been in protein labeling. Initially, a CoA molecule derivatized on the thiol group with a maleimide-linked reporter group was employed as a substrate for a phosphopantetheinyl transferase. The reporter-modified phosphopantetheine moiety was thereby enzymatically transferred from the CoA derivative 35 to the apoform of the carrier protein of polyketide synthases and nonribosomal peptide synthetases to form the labeled holo-carrier protein analog 36 (Fig. 7) (64). The reporter group was either a fluorophore, for fluorescent visualization of the reporter-modified protein or biotin or other affinity tag to facilitate isolation of the labeled protein. In subsequent developments of this technology, it has been applied to the labeling of fusion proteins between the relatively small carrier protein and another protein, the fused carrier protein domain thereby directing labeling to the protein of interest (65). This methodology has also been applied to the labeling of fusion proteins on cell surfaces (66). More recently, it has been demonstrated that the CoA-reporter derivatives 35 can be prepared from synthetic pantetheine-reporter conjugates 32 using the pantothenate kinase, phosphopantetheine adenylyltransferase, and dephospho-CoA kinase enzymes of CoA biosynthesis (67). The enzymes are quite promiscuous in converting analogs containing only the pantoic amide moiety of the natural pantothenate or pantotheine substrate, generally with a flexible tether between the amide and the labeling group of interest. Pantoic amide analogs containing reactive azide, alkyne, and ketone functionality as well as biotin and fluorescent labels have been employed to incorporate the functionality into the protein substrate of the phosphopantetheinyl transferase. The significance of the enzymatic generation of the CoA analog substrates for the phosphopantetheinyl transferase is that simple cell-permeable pantoic amide analogs can be processed all the way to the labeled carrier protein or fusion protein thereof in vivo. A set of bioorthogonal pantoic amide analogs have been developed for use in this in vivo protein tagging method (17). Other recent work has demonstrated that a simple 11-residue peptide incorporated into the targeted protein can be recognized by the phosphopantotheinyl transferase Sfp from Bacillus subtilis (68), which promises to be a very general and versatile method for in vivo protein tagging and labeling.

 

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See Also

Enzyme Catalysis, Chemical Strategies for

Enzyme Cofactors, Chemistry of

Vitamins, Chemistry of

Small Molecules to Elucidate Biological Function