Chapter 44

Biosynthesis of Pantothenic Acid and Coenzyme A

SUZANNE JACKOWSKI

INTRODUCTION

Pantothenic acid is one of the B complex of vitamins and is a nutritional requirement in mammals. Bacteria divert amino acids and their biosynthetic intermediates to produce pantothenate, which is used primarily for the synthesis of coenzyme A (CoA) and acyl carrier protein (ACP), the two the predominant acyl group carriers in cells. The acyl moieties are attached to the terminal sulfhydryl of the 4'-phosphopantetheine prosthetic groups of these two cofactors. These compounds are essential cofactors in all cells and participate in over 100 different reactions in intermediary metabolism (2). Short-chain CoA thioesters (acetyl-CoA, succinyl-CoA, etc.) are the most abundant components of the CoA pool and are critical intermediates in carbon source metabolism (see chapter 16). In Escherichia coli and Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium), CoA thioesters of long chain fatty acids are the intermediates in β oxidation (see chapter 21) and the incorporation of exogenous fatty acids into phospholipids (see chapter 37), and they also regulate the DNA binding of a transcription factor that represses the enzymes of β oxidation and activates the transcription of fabA, a key enzyme in fatty acid biosynthesis (13, 22, 23). In contrast, ACP thioesters carry the acyl group intermediates in de novo fatty acid biosynthesis (see chapter 37). In addition, CoA and its thioesters modulate the activity of several key enzymes in intermediary metabolism (2). 4'-Phosphopantetheine prosthetic groups are also found in other enzyme systems, such as the entF gene product involved in serine activation in the biosynthesis of E. coli siderophore enterobactin (59). Most of what is known about the biochemistry and genetics of the enzymes involved in the formation of pantothenate and CoA has been derived from research with E. coli and S. typhimurium.

BIOSYNTHESIS OF PANTOTHENATE AND ITS PRECURSORS

α-Ketopantoate Biosynthesis

The first step in the biosynthesis of d-pantoic acid is the transfer of a methyl group from 5,10-methylenetetrahydrofolate to α-ketoisovaleric acid by α-ketopantoate hydroxymethyltransferase (EC 2.1.2.11) (Fig. 1). The idea that α-ketoisovalerate, an intermediate in the biosynthesis of valine, is also required for pantoate synthesis was first suggested by Maas and Vogel (39) on the basis of the finding that E. coli could convert α-ketoisovalerate to pantoate whereas a pantoate-requiring mutant could not. Metabolic labeling experiments show that the conversion of α-ketoisovalerate to pantothenate proceeds stereospecifically, with inversion of the configuration at the C-3 carbon of a-ketoisovalerate (1). The analysis of mutants lacking a,β-dihydroxyisovalerate dehydrase (ilvD), transaminase B (ilvE), and transaminase C (avtA) shows that the sole pathway to pantothenate is from α-ketoisovalerate (76). Snell and coworkers (52, 53, 67) purified and biochemically characterized α-ketopantoate hydroxylmethytransferase. They found that pantothenate (>500 μM) and CoA (>1 mM) were allosteric inhibitors of transferase activity and suggested that this may be of regulatory significance in pantothenate synthesis. However, these concentrations of inhibitors are much higher than the estimated concentrations of the compounds in vivo (70). McIntosh et al. (42) proposed that α-ketopantoate could be formed from α-ketoisovalerate and formic acid. Although this reaction is catalyzed by E. coli extracts, it is clear that this enzyme does not play a role in α-ketopantoate synthesis in vivo, since mutants that require α-ketopantoate for growth retain lactone reductase activity but lack the 5,10-methylenetetrahydrofolate-dependent hydroxymethyltransferase (67).

Fig. 1Pathway for the biosynthesis of pantothenic acid. Three enzymatic steps are required for the de novo formation of pantothenate. ?-Alanine is formed from aspartate by aspartate-1-decarboxylase, the product of the panD gene. Pantoate formation begins with the transfer of a methyl group to ?-ketoisovalerate by ketopantoate hydroxymethyltransferase (the panB gene product) followed by reduction by ketopantoate reductase (the panE gene product). Pantothenate is then formed by the ATP-dependent condensation of ?-alanine and pantoate by pantothenate synthetase (the panC gene product). Pantothenate is then either used for CoA biosynthesis or exported from the cell.

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α-Ketopantoate hydroxymethyltransferase is the product of the panB, gene which was cloned and sequenced by Jones et al. (31). The gene contains an open reading frame of 792 bp preceded by its own promoter. The subunit molecular weight predicted from the gene sequence is 28,179, and electrospray mass spectroscopy of the purified protein yielded a subunit molecular weight of 28,178 ± 5. The molecular weight estimated from gel filtration chromatography is 174,000, indicating that the native protein exists as a hexamer. The panB gene is located at min 3.1 of the E. coli chromosome in the panB-panD-panC gene cluster (9). The genetic mapping suggests that these genes may be organized in an operon. Northern (RNA) analysis with a specific panB probe detects a 1.9-kb mRNA (31). This mRNA is larger than predicted from the 792-bp panB gene; however, definitive information on the molecular structure of the panD and panC genes will be required to determine if one, both, or neither of these genes is cotranscribed with panB.

In early work, Demerec et al. (12) isolated a mutant of S. typhimurium that responded to α-ketoisovalerate and valine as well as ketopantoate, pantoate, and pantothenate and thus proposed that the mutation, designated panA, confers defective synthesis of α-ketoisovalerate. Cronan pointed out that this hypothesis would require the strains to be auxotrophic for valine and leucine in addition to pantothenate (8). Cronan et al. (9) then investigated the biochemical basis for the panA mutation and showed that extracts from the panA strain were deficient in α-ketopantoate hydroxymethyltransferase. Therefore, the panA lesion is an allele of the panB gene and not a separate locus.

Pantoate Biosynthesis

d-Pantoate is synthesized from α-ketopantoate by α-ketopantoate reductase, the panE gene product (EC 1.1.1.169) (Fig. 1). The first evidence indicating that α-ketopantoate is the precursor to pantoate was provided by Lansford and Shive (33), who showed that one class of pantothenate auxotrophs could be supplemented with α-ketopantoate as well as pantoate. The reductase was biochemically characterized by Wilken et al. (77), who reported the presence of reductase activity in E. coli and in Saccharomyces cerevisiae. The reductase is a B-specific enzyme which transfers tritium from [4B-³H]NADPH into pantoate. Primerano and Burns (55, 56) were responsible for isolating mutants in α-ketopantoate reductase and clarifying the relationship between pantoate and branched-chain amino acid biosynthesis. The key point is that acetohydroxy acid isomeroreductase (EC 1.1.1.86), a reductase involved in the biosynthesis of isoleucine and valine (the ilvC gene product), is also capable of catalyzing the reduction of α-ketopantoate. Thus, panE mutants do not require pantoate when the ilvC gene is abundantly expressed, but when ilvC expression is low, panE mutants require pantoate for growth (56). Recognition of this fact allowed panE mutants to be isolated beginning with S. typhimurium ilvC mutants (56). The panE gene is not located close to the panBDC cluster (56), and a mutation in E. coli presumed to lead to a defect in α-ketopantoate reductase lies at min 87 close to metB (40). However, these data are not definitive and the precise location of the panE gene remains to be determined. The best-characterized α-ketopantoic acid reductase was purified and crystallized from Pseudomonas maltophilia 845 by Shimizu et al. (60). The purified enzyme has a subunit molecular weight of 30,500 and exists in solution as an oligomer containing three to five subunits. These investigators also isolated P. maltophilia pantothenate auxotrophs and showed that these strains were deficient in α-ketopantoate reductase but had normal levels of 2-acetolactate isomeroreductase, 2-keto-3-hydroxyisovalerate reductase, and ketopantoyl lactone reductase. It is important to understand that ketopantoate reductase is distinct from the ketopantoyl lactone reductases (EC 1.1.1.168) (77, 78). The physiological function of the ketopantoyl lactone reductases is unknown, although it is clear that they do not participate in pantothenate biosynthesis since mutants that require pantoate for growth still possess wild-type levels of ketopantoyl lactone reductases (60).

β-Alanine Biosynthesis

Aspartate was first suggested as the precursor to β-alanine on the basis of the conversion of aspartate to β-alanine by intact cells (10, 75). Williamson and Brown (80) and Cronan (8) independently characterized an l-aspartate-1-decarboxylase activity from E. coli that converts aspartate to CO₂ and β-alanine (EC 4.1.1.11) (Fig. 1). The decarboxylase has a molecular weight of 58,000 as determined by gel filtration chromatography, but the complicated pattern of elution observed on ion-exchange chromatography and sodium dodecyl sulfate (SDS)-gel electrophoresis (79, 80) means that conclusions concerning the subunit structure of the enzyme are tenuous at present. Pyridoxyl phosphate is the common cofactor found in amino acid decarboxylases; however, the active carbonyl moiety in the aspartate decarboxylase reaction is provided by covalently bound pyruvate (79). Whether the pyruvate moiety is formed autocatalytically or another "activating" enzyme is responsible for the conversion of the proenzyme to the active form remains to be determined. Autocatalytic activation of the proenzyme seems most likely, since this mechanism has been established for other amino acid decarboxylases containing pyruvate (58). The importance of this enzyme in CoA biosynthesis was confirmed by the finding that mutants (panD) that require β-alanine for growth lack asparate-1-decarboxylase activity (8, 80). The inhibition of decarboxylase activity by d-serine, β-hydroxyaspartic acid, or l-cysteic acid is consistent with the interference of pantothenic acid synthesis by these compounds in intact bacteria (7, 15, 16, 21, 37, 61). A second pathway for the synthesis of β-alanine from dihydrouracil proposed by Slotnick and Weinfeld (62, 63) does not occur, since Cronan (8) showed that β-alanine auxotrophs (panD mutants) will not grow on dihydrouracil. The panD gene is located at min 3.1 of the E. coli chromosome and is part of a three-gene cluster with a clockwise gene order of panB-panC-panD (9). The panD locus in S. typhimurium was first reported by Ortega et al. (47) to be located at min 89, separate from the panB and panC genes; however, Primerano and Burns (56) have more recently isolated additional S. typhimurium β-alanine auxotrophs with lesions at min 4.5 adjacent to panB and panC. It is not clear whether mapping errors are responsible for these different results or whether a functional aspartate-1-decarboxylase is composed of heterologous subunits that are produced by different genes. It is also possible that the auxotrophs isolated by the Burns laboratory are actually panC mutants with synthetases exhibiting a high K_m for β-alanine. The molecular analysis of the panD gene and its product is the next important step in understanding the structure and function of the decarboxylase.

Pantothenate Biosynthesis

Pantothenate synthetase (EC 6.3.2.1) catalyzes the ATP-dependent condensation of pantoate with β-alanine (Fig. 1). Pantothenate synthetase has been extensively studied first in partially purified forms (32, 36, 38, 48) and then in a homogeneous form (43, 44, 45). The stoichiometric formation of AMP and pyrophosphate (44; W. K. Maas, Fed. Proc. 15:305, 1956) and the kinetic analysis of pantothenate synthetase (44) are consistent with a bi uni bi ping-pong mechanism involving the formation of an enzyme:pantoyl-AMP intermediate according to the following scheme:

Pantoate + ATP + enzyme ↔ [enzyme:pantoyl-AMP] + PP_i

[Enzyme:pantoyl-AMP] + β-alanine → pantothenate + AMP + enzyme

Pantothenate synthetase exists as a homotetramer, as judged from the molecular weight of 70,000 calculated from sedimentation equilibrium ultracentrifugation data and the subunit molecular weight of 18,000 calculated from SDS-gel electrophoresis and amino acid composition (43, 44, 45). The genetic and biochemical analyses of both E. coli and S. typhimurium have established pantothenate synthetase as the product of the panC gene (9, 36). In E. coli, panC is located at min 3.1 min on the chromosome, and the clockwise gene order is panB-panC-panD (9). Pantothenate synthetase activity is not tightly regulated in vivo since E. coli secretes into the medium most of the pantothenate synthesized (11, 26, 37). The significance of pantothenate overproduction in bacteria is unknown, but the fact that E. coli excretes a copious amount of pantothenate points to a role for intestinal flora in providing this vitamin to the mammalian host.

Pantothenate Transport

Bacteria are capable of the bidirectional transmembrane movement of pantothenate (Fig. 2). The best-characterized transport system is the high-affinity pantothenate permease of E. coli that catalyzes the concentrative uptake of pantothenate by a sodium ion cotransport mechanism (46, 72). Pantothenate permease belongs to the group of inner membrane permeases that catalyze active cation-dependent symport (chapter 74). A similar transport system is present in S. typhimurium (S. D. Dunn and E. E. Snell, J. Supramol. Struct. 6:136, 1977). Since E. coli can synthesize pantothentate, the pantothenate uptake system is dispensable; however, in bacteria that lack a pantothenate biosynthetic pathway (e.g., Lactobacillus spp.), pantothenate permease activity is required for growth. Jackowski and Alix (25) have cloned the E. coli gene for the permease and determined its sequence. The permease has a predicted molecular weight of 51,992, and sequence analysis predicts that pantothenate permease is an integral membrane protein with 12 hydrophobic membrane-spanning domains connected by short hydrophilic sequences. This predicted topological profile is similar to those of other membrane carriers that catalyze cation-dependent symport. The panF gene is located at min 72 of the chromosome (71) and is cotranscribed with prmA, a protein responsible for methylation of ribosomal proteins (74). The physiological significance, if any, of the coordinate transcription of these two genes is unknown. In contrast, almost nothing is known about the biochemistry or molecular biology of the pantothenate efflux system. However, efflux is not affected in panF mutants (71); therefore, a separate, uncharacterized transport system is responsible for the expulsion of pantothenate from cells. The efflux system may play a role in the kinetic control of pantothenate phosphorylation by ensuring that the intracellular concentration of pantothenate remains low.

Fig. 2Pathway for CoA biosynthesis and the addition of the prosthetic group to ACP. Active uptake of pantothenate by a sodium-dependent permease (the panF gene product) is an alternate route to intracellular pantothenate, which is then either used for CoA biosynthesis or exported from the cells by a separate transport system. Pantothenate kinase (the coaA gene product) is the first, and most highly regulated, step in CoA biosynthesis and is regulated by feedback inhibition by CoA and its thioesters. Cysteine is then added to 4?-phosphopantothenate to generate 4?-phosphopantothenoylcysteine, which is then decarboxylated to yield 4?-phosphopantetheine. 4?-Phosphopantetheine adenylyltransferase is a secondary regulatory point and is responsible for the formation of dephospho-CoA, which is then phosphorylated on the 3?-hydroxyl group to yield CoA. CoA is then used as the 4?-phosphopantetheine donor in the synthesis of ACP from apo-ACP catalyzed by ACP synthase (the acpS gene product). The 4?-phosphopantetheine prosthetic group of ACP undergoes rapid metabolic turnover, generating apo-ACP and 4?-phosphopantetheine. The direct degradation of CoA to 4?-phosphopantetheine occurs during abrupt metabolic transitions. 4?-Phospho- pantetheine is either reused for CoA synthesis or excreted from the cell. 4?-Phosphopantetheine accumulates in the growth medium since E. coli does not have an uptake system for this intermediate.

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CoA BIOSYNTHESIS

Pantothenate Phosphorylation

Pantothenate kinase (EC 2.7.1.33) catalyzes the first and most highly regulated step in the biosynthesis of CoA (Fig. 2). Originally it was thought that the first phosphorylated intermediate in the pathway was 4'-phosphopantetheine; however, this point was reinvestigated in 1958 by Brown (6), who convincingly showed that 4'-phosphopantothenate was synthesized from ATP and pantothenate and that this intermediate was required for the subsequent reactions in the CoA biosynthetic pathway. Temperature-sensitive pantothenate kinase (coaA) mutants were isolated by Dunn and Snell in S. typhimurium (14) and subsequently by Vallari and Rock in E. coli (73). Actually, the first temperature-sensitive coaA mutants were isolated in 1966; however, the biochemical defect was unknown, and the mutated gene was termed rts (19). The rts and coaA mutations are alleles of the same gene (64), as indicated by the fact that the nucleotide sequences of rts and coaA are identical (20, 65). The coaA mutants cannot be supplemented since E. coli does not incorporate extracellular 4'-phosphopantetheine or CoA. The coaA gene is located at min 90 of the E. coli chromosome between the birA and thrU genes (65). The coaA gene has its own promoter and produces a 1.1-kb transcript. The utilization of either of two translational initiation sites produces two pantothenate kinase proteins that differ in eight amino acids at the amino terminus. The poor homology of the coaA promoter region to consensus E. coli promoter sequences and the low frequency of optimal codon usage are consistent with a low level of pantothenate kinase expression (65).

Jackowski and Rock (26) proposed pantothenate kinase as a major rate-controlling step in CoA biosynthesis on the basis of the copious excretion of pantothenate from E. coli (11, 26, 37). Nonesterified CoA was five times more potent than CoA thioesters in inhibiting the enzyme in extracts from E. coli. CoA is a competitive inhibitor with respect to ATP, thus providing a mechanism to coordinate CoA production with the energy state of the cell. There are considerable differences in the size and composition of the CoA pool in E. coli cells grown in different carbon sources (70). A shift from glucose to acetate as the carbon source resulted in an increase in the nonesterified CoA/acetyl-CoA ratio from 0.7 to 4.3. This alteration in the CoA pool composition was associated with the selective inhibition of pantothenate phosphorylation, consistent with nonesterified CoA being the most potent inhibitor of pantothenate kinase in vivo. E. coli mutants which possessed a pantothenate kinase activity in crude extracts that was refractory to feedback inhibition by CoA were isolated (69). Strains harboring this mutation [coaA16(Fr)] had CoA levels that were significantly (>2-fold) higher than in strains containing the wild-type kinase. Corroboration of the conclusion that modulation of pantothenate kinase activity by feedback regulation is the critical factor controlling the intracellular CoA concentration comes from studies of the effects of pantothenate kinase overexpression on the size of the CoA pool (65). Strains expressing 76-fold more wild-type kinase exhibited only a 2.7-fold increase in the steady-state CoA level. Pantothenate kinase was purified to homogeneity from overexpressing strains and shown to be a homodimer consisting of 36-kDa subunits. The purified enzyme exhibits cooperative binding of ATP which is competitively inhibited by CoA. Cooperative ATP binding made determination of the kinetic mechanism complicated; to overcome problematic interpretation of the kinetic data, intragenic complementation was used to produce a chimeric heterodimer between a wild-type and an inactive subunit. The inactive subunit was expressed by a coaA plasmid construct in which the codon for the critical lysine in the ATP binding site was mutated to encode a methionine residue. Kinetic analysis of these chimeric molecules indicated the absence of cooperative ATP binding and revealed that the kinase reaction proceeds by a sequential mechanism, with ATP as the first substrate interacting with the protein (66). Taken together, these data lead to the conclusion that pantothenate kinase activity is effectively regulated by feedback inhibition primarily by the concentration of nonesterified CoA and secondarily by the size of the CoA thioester pool.

Formation of 4'-Phosphopantetheine

4'-Phosphopantetheine is formed in two enzymatic steps (Fig. 2). First, 4'-phosphopantothenate is condensed with cysteine by 4'-phosphopantethenoyl-1-cysteine synthetase (EC 6.3.2.5) to form 4'-phosphopantenoylcysteine, which is then decarboxylated by 4'-phosphopantothenoylcysteine decarboxylase (EC 4.1.1.36) to generate 4'-phosphopantetheine. Basically nothing is known about the biochemistry or genetics of these two enzymes in E. coli or S. typhimurium beyond the demonstration of the activities in crude extracts by Brown (5). Neither 4'-phosphopantothenate nor 4'-phosphopantethenoylcysteine is detected in vivo (26) owing to the rapid conversion of both to 4'-phosphopantetheine. Methods for the positive selection for conditional mutants affected in these two enzymes need to be developed to advance investigation of this portion of the pathway.

Conversion of 4'-Phosphopantetheine to CoA

There are two enzymatic steps that convert 4-phosphopantetheine to CoA (3, 24; Fig. 2). First, ATP:4'-phosphopantetheine adenylyltransferase (EC 2.7.7.3) transfers the AMP moiety to 4'-phosphopantetheine, with the release of PP_i to form dephospho-CoA. Next, a phosphate group is added to the 3'-hydroxyl of dephospho-CoA by dephospho-CoA kinase (EC 2.7.1.24). Again, little is known about the biochemistry or genetics of these two enzymes in either E. coli or S. typhimurium. In mammals, these two enzymes are copurified and exist as a bifunctional protein that is designated CoA synthase (81). However, these two activities have been separated by chromatographic fractionation of Brevibacterium ammoniagenes extracts, suggesting that individual enzymes catalyze the two steps in bacteria (41). Metabolic labeling experiments detect both intracellular and extracellular 4'-phosphopantetheine, suggesting that 4'-phosphopantetheine adenylyltransferase is a secondary regulatory point in CoA biosynthesis (26, 69). Double-label experiments indicate that extracellular 4'-phosphopantetheine is derived from the degradation of ACP (see below), and reutilization of this intermediate is regulated at the adenylyltransferase step (29). Excretion of 4'-phosphopantetheine is an irreversible mechanism for reduction of the intracellular CoA and ACP content since E. coli is unable to assimilate exogenous 4'-phosphopantetheine into CoA (29). Understanding the biochemical mechanisms that regulate the activity of 4'-phosphopantetheine adenylyltransferase will be necessary to establish the role of this enzyme in controlling the intracellular CoA concentration.

CoA METABOLISM

Composition of the Intracellular CoA Pool

The composition of the intracellular CoA pool has been examined in E. coli by using panD mutants to label CoA specifically and then estimating individual CoA thioesters by high-pressure liquid chromatography (70). The size and composition of the CoA pool vary depending on the carbon source (Table 1). The CoA pool is highest in cells growing on glucose, and acetyl-CoA is the predominant thioester. In contrast, the amount of total CoA is much lower in cells growing on casein hydrolysate as the carbon source, indicating that amino acid biosynthesis requires an elevated CoA pool, particularly in the form of acetyl-CoA. Consistent with this interpretation, when pantothenate auxotrophs growing on glucose minimal medium are deprived of pantothenate, the succinyl-CoA pool becomes limiting for amino acid and protein synthesis, leading to growth arrest (30). Alternatively, growth on glucose may lead to acetate accumulation, which, in turn, may determine the size of the acetyl-CoA pool. The latter explanation is supported by the observation that the acetyl-CoA pool is rapidly reduced >30% upon withdrawal of supplement from an acetate auxotroph (69). Reports of oxidized CoA derivatives (e.g., CoA:glutathione mixed disulfides [34, 35]) are likely due to oxidation of the samples prior to analysis, and there is no evidence that these derivatives occur in vivo.

Table 1Intracellular CoA pool composition in E. colia

Role of CoA in ACP Biosynthesis and Turnover

CoA serves as a precursor to ACP via the transfer of its 4'-phosphopantetheine moiety to apo-ACP (the product of the acpP gene [57]) by holo-ACP synthase (CoA:apo-ACP pantetheine phosphotransferase; EC 2.7.8.7) (17, 54; Fig. 2). ACP synthase is the product of the acpS gene, and conditional mutants were isolated by Polacco and Cronan (49). The enzyme has a K_m defect in CoA binding such that cells can grow on high concentrations of extracellular pantothenate but fail to grow on lower pantothenate concentrations that are normally able to support the growth of E. coli. Cells normally contain undetectable levels of apo-ACP; however, in acpS mutants grown under nonpermissive conditions, the major portion of ACP is in the apo form (27). Metabolic labeling following the starvation of pantothenate auxotrophs shows that the level of active ACP is maintained at the expense of CoA (4, 26, 29). The ACP prosthetic group is removed by the action of ACP phosphodiesterase (EC 3.1.4.14) (68), a heat-stable, 25-kDa protein (18). ACP prosthetic group turnover is rapid compared with the stability of the protein moiety (28, 50, 51), thus generating a futile cycle of ACP synthesis and degradation. The physiological significance of this cycle remains a mystery, although it seems unlikely to be related to the regulation of fatty acid biosynthesis (27). The 4'-phosphopantetheine released from ACP can be either reincorporated into CoA or excreted from the cell (29). When the CoA level is low, however, turnover of the ACP prosthetic group is faster and excretion of 4'-phosphopantetheine is minimal, suggesting that the cycle participates in the metabolic maintenance of a basal CoA concentration (28). 4'-Phosphopantetheine excretion is an irreversible mechanism to decrease the CoA pool, since E. coli is unable to incorporate extracellular 4'-phosphopantetheine into CoA (29).

CoA Degradation

A third potential source of 4'-phosphopantetheine is by the direct degradation of CoA by a phosphodiesterase. CoA degradation occurs when the levels of acetyl-CoA fall, leading to a concomitant increase in the nonesterified CoA pool (69). CoA hydrolysis following this type of abrupt physiological transition appears not to involve ACP prothetic group turnover but to occur by the direct degradation of CoA. The physiological significance of this process is unknown, but the maintenance of a proper acetyl-CoA/CoA ratio may be of regulatory significance in controlling the activities of other enzymes in intermediary metabolism. This is the only reported instance of CoA degradation in E. coli, and the enzyme(s) involved in this process remains to be identified and characterized.