Chapter 16

Tricarboxylic Acid Cycle and Glyoxylate Bypass

JOHN E. CRONAN, JR., and DAVID LaPORTE

INTRODUCTION

The tricarboxylic acid (TCA) cycle (Fig. 1) plays two essential roles in metabolism (3). First, the cycle is responsible for the total oxidation of acetyl coenzyme A (CoA), which is derived mainly from the pyruvate produced by glycolysis. Second, TCA cycle intermediates are required in the biosynthesis of several amino acids as well as for heme biosynthesis. Although the TCA cycle has long been considered a "housekeeping" pathway, recent evidence indicates that in Escherichia coli and Salmonella typhimurium the pathway is inducible (32, 42, 43). Inducibility was demonstrated by enzymatic assays for some gene products. In contrast, other TCA cycle enzymes appeared to be expressed constitutively (28). Subsequent work demonstrated that expression of this latter group of enzymes is also inducible. The appearance of constitutivity had resulted from differential regulation of two or more genes (31, 32). For example, the anaerobic repression of succinate dehydrogenase was masked by the anaerobic induction of fumarate reductase (31). Similarly, regulation of the aerobic fumarase, FumA, was masked by the anaerobic FumB and the unregulated FumC (98).

Fig. 1TCA cycle and glyoxylate bypass. The glyoxylate bypass is composed of isocitrate lyase and malate synthase. IDH and IDH-P indicate the dephosphorylated and phosphorylated forms of IDH, respectively.

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The expression levels of the TCA cycle enzymes respond primarily to the presence of oxygen (3, 83) and to the carbon source (3, 28). In E. coli, the full TCA cycle (Fig. 1) is seen only during aerobic growth on acetate or fatty acids (3, 68). These culture conditions give the highest levels of TCA cycle enzymes, and the cycle provides all the energy and reducing potential needed to support growth. However, growth on acetate or fatty acids requires induction and function of an anaplerotic pathway, the glyoxylate shunt (45, 46), to replenish the dicarboxylic acid intermediates consumed in amino acid biosynthesis. This will also be discussed. It should be noted that the great bulk of recent work on the TCA cycle is that of Guest and coworkers, and these investigators have written several recent reviews (31, 32, 63). Due to space limitations we shall not repeat the references to the original literature readily found in these reviews, but will instead reference the most relevant review. In contrast, most of the voluminous literature on the enzymology of the TCA cycle enzymes has not been reviewed recently, and if no review is available, we shall provide a recent reference as an entree into the literature.

Under anaerobic conditions, the TCA cycle can no longer provide energy and instead functions as two biosynthetic pathways, a reductive pathway that produces succinyl-CoA acid and an oxidative pathway producing 2-ketoglutarate (Fig.2). These pathways fail to form a cycle because 2-ketoglutarate dehydrogenase is virtually absent during anaerobic growth (3, 43, 82) (synthesis of this enzyme is the most severely repressed of all the TCA cycle enzymes). Under these conditions, the levels of the other enzyme activities (and enzyme proteins) are much lower (10- to 20-fold) than those found during aerobic growth. This reduction in expression is appropriate since a much lower flux would be required when only biosynthetic functions are served (3, 28, 32, 43, 82).

Fig. 2Function of the TCA cycle in anaerobic growth. The oxidative branch is to the right and the reductive branch is to the left.

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During anaerobic growth, several other enzymes are induced to augment the biosynthetic pathways formed by the TCA cycle enzymes. For example, fumarate reductase replaces succinate dehydrogenase to allow reductive production of succinyl-CoA (as well as providing an anaerobic respiratory pathway). Aspartase is induced to assist (together with the constitutive aspartate aminotransferase) in the conversion of oxaloacetate to fumarate. Furthermore, some carbon does flow between the branches by the action of the glyoxylate cycle enzyme isocitrate lyase, which converts isocitrate to succinate and glyoxylate (14). The action of isocitrate lyase under these conditions is demonstrated by the finding that anaerobically grown cells display a succinate requirement only when both isocitrate lyase and fumarate reductase are inactivated by mutation (14).

The branched, biosynthetic form of the TCA cycle is also used by aerobic cultures growing on glucose (Fig. 2). The full TCA cycle is not required under these conditions because the bulk of energy is derived from glycolysis (3).

The TCA cycle enzymes may also be subject to end product repression by amino acids (28, 32). Repression by amino acids would seem advantageous in the branched mode of the cycle, and data suggesting a small (two- to fourfold) decrease in TCA cycle enzyme levels upon addition of amino acid mixtures to aerobic glucose-grown cultures have been reported (28). However, other data indicate that addition of glutamate (the major TCA cycle-derived amino acid) to such glucose cultures gave increased enzyme activity (3). These conflicting reports may result from subtle differences in growth conditions. For example, aerobic glucose-grown cultures of low cell densities have much lower 2-ketoglutarate dehydrogenase activities than do more dense cultures (3). This finding has been attributed to acetate accumulation in the more dense cultures (3). When the TCA cycle must function as the primary source of energy, amino acid repression (if it occurs) would probably be a disadvantage. Consistent with this prediction, very similar levels of 2-ketoglutarate dehydrogenase activity are seen when cultures are grown on either acetate or glutamate as sole carbon source (3).

GENE-ENZYME RELATIONSHIPS IN THE TCA CYCLE

Most of the E. coli genes which encode the TCA cycle enzymes and the major auxiliary enzymes have been cloned, sequenced, and placed on the genetic and physical maps (32, 63) (Table 1). One 13-kbp chromosomal segment contains nine TCA cycle genes which encode the subunits of four of the cycle enzymes, whereas the genes encoding the remaining enzymes are scattered around the chromosome. In general the genes encoding the subunits of multisubunit complexes are adjacent and cotranscribed, the exception being the gene (lpd) encoding the E3 subunit of 2-ketoglutarate dehydrogenase, which can be either cotranscribed with the genes (aceEF) encoding the subunits of pyruvate dehydrogenase (the E3 subunit is common to both 2-ketoacid dehydrogenases) or transcribed separately (63, 74). Transcriptional mapping of the TCA cycle gene cluster has provided no strong rationale for clustering (63, 83). Indeed the gltA (citrate synthase) gene is transcribed in the direction opposite to the other TCA cycle genes (63). Most genes within this cluster are transcribed from two different promoters, and those transcripts that encode subunits of two TCA cycle enzymes (e.g., sucAB and sucCD) provide the only rationale for clustering (63). However, since the sucCD gene products are required for anaerobic growth (60) whereas those encoded by sucAB are not, there must be a second sucCD promoter (63).

Table 1Genes and enzymes.

Many of the TCA cycle genes are named for the enzyme encoded, and others are named for the TCA cycle intermediate needed to bypass the enzyme deficiency. Some of these gene names illustrate the dual functions of the TCA cycle. For example, the citrate synthase gene is called gltA because mutants lacking the enzyme require 2-ketoglutarate, which is readily supplied as glutamate or proline. Similarly, mutations in sucAB, which encode two of the three 2-ketoglutarate dehydrogenase subunits, result in a requirement for succinate for aerobic growth on glucose (14, 15). However, some of the gene designations seem both confusing and misleading. The sucCD genes encode the two subunits of succinyl-CoA synthetase, and mutants defective in this enzyme are unable to utilize succinate as a source of succinyl-CoA when grown anaerobically (60). Therefore, the suc designation has opposite meanings in the sucAB and sucCD cases. Moreover, the use of consecutive letters implies that the gene products are closely related, perhaps being subunits of a single enzyme complex (as is the case for the adjacent operon, sdhABCD) rather than two successive enzymes of the pathway. We suggest that stkAB (for succinate thiokinase, a more proper name for succinyl-CoA synthase) would be a more fitting designation for sucCD.

In many cases, the deduced amino acid sequences of the E.coli TCA cycle enzymes provided the first complete primary structures of the corresponding enzymes. Subsequent isolations of the TCA cycle genes from other organisms have generally given deduced amino acid sequences that strongly resemble those of the E. coli proteins.

MUTANTS DEFECTIVE IN THE ENZYMES OF THE TCA CYCLE

Mutants lacking any of the TCA cycle enzymes have a common phenotype: all fail to grow with acetate as sole carbon and energy source. Some TCA cycle mutants (sucAB, mdh, sdhCDAB, and fumA) grow normally on glucose minimal medium under anaerobiosis, and this growth is attributed either to a lack of function of the enzyme in the branched mode or to other enzymes that supply the missing biosynthetic intermediates. A caveat to some of these findings is that much of this work was done before null mutants (e.g., transposon insertions) in bacterial genes could be readily isolated, and some of the mutants tested may have retained activity undetectable by enzymatic assay. The enzymes involved in the oxidative side of the branched pathway, citrate synthase, aconitase, and isocitrate dehydrogenase (IDH), are responsible for glutamate synthesis and thus should be essential for both aerobic and anaerobic growth on glucose minimal media. Mutants lacking citrate synthase or IDH are known to be glutamate auxotrophs under aerobic conditions, but the question of auxotrophy under anaerobic conditions does not appear to have been addressed. Therefore, one of us (J.E.C.) tested two different gltA null mutants (both made by in vitro construction followed by recombination into the chromosome) and found that under anaerobiosis neither mutant strain grew on glucose unless supplemented with glutamate.

The recent reports of the acn gene isolation (72) and sequence (73) mean that genes encoding each of the TCA cycle enzymes have been cloned and sequenced. Most of these genes were isolated by complementation of mutants having lesions within the target genes (63). Other TCA cycle genes were isolated by sequencing out from known TCA cycle genes (63). In some cases, reverse genetic approaches were required. These approaches included screening with oligonucleotides which were based on amino acid sequence data, as well as screening with antibodies (72, 88). Although these genes were cloned and sequenced, strains carrying mutations in certain of these genes have not yet been isolated. In these cases, there is no in vivo evidence that the gene isolated is solely responsible for a given TCA cycle reaction. Interpretation of enzyme assay data is also complicated for those enzymes which contain Fe-S centers, since these activities are often labile in conventional cell-free extracts. The recent demonstration of a second aconitase in E. coli illustrates the complications caused by unstable enzymes (29).

THE ENZYMES OF THE TCA CYCLE

Citrate Synthase

E. coli citrate synthase is a hexamer of the gltA-encoded protein (94; for review, see reference 95). The allosteric behavior of this enzyme is typical of the citrate synthases of gram-negative bacteria and distinct from the enzymes of other bacteria and mammalian mitochondria (94). E. coli citrate synthase is inhibited by 2-ketoglutarate and activated by NADH, properties consistent with the function of the TCA cycle in the branched and energy-producing modes, respectively (94). The allosteric properties of this enzyme are complex and remain under active investigation (70). E. coli citrate synthase has not yet been crystallized, and this has slowed detailed study of the enzyme, although good progress on the active site has been made by use of the known crystal structures of the vertebrate enzymes (70).

Use of controlled overproduction of citrate synthase shows that this enzyme catalyzes the rate-limiting step of the TCA cycle when E. coli is grown on acetate, but is normally made in functional excess in aerobic cultures grown on glucose (91). However, altered production of citrate synthase could also effect the phosphorylation of IDH (see below), and thus it would be of interest to repeat the glucose growth experiments in an aceK null mutant.

There is evidence that E. coli can synthesize an enzyme other than GltA that catalyzes the citrate synthase reaction. Patton et al. (69) have reported that some revertants of gltA6 strains able to grow without glutamate possess a new citrate synthase that is readily distinguished from GltA by physical properties, N-terminal sequence, and immunological determinants. It seems very likely that this enzyme is encoded by a gene which is not expressed in wild-type strains. Similar mutants were isolated previously (16), and the mutant gene was located close to gltA on the genetic map. It should be noted that the insensitivity of the citrate synthase of this mutant to 2-ketoglutarate and NADH and the excretion of a glutamatelike metabolite were taken as evidence for the importance of these allosteric effectors in GltA action (16). However, since the revertant activity is not encoded by gltA, this interpretation is invalid.

Aconitase

The aconitase of E. coli has only recently been purified to homogeneity and shown to be a dimeric protein, encoded by the acn gene, which contains a labile iron-sulfur cluster (73). A null mutant lacking this activity has been reported but grows normally, indicating the presence of another aconitase (29). It should be noted that a prior report of the isolation of the acn gene by hybridization to the Bacillus subtilis citB gene was in error, although strains carrying the cloned E. coli DNA segment markedly overproduced aconitase activity (96). This curious result deserves further investigation. There is a possibility that aconitase may play a role in sensing intracellular iron concentrations (72, 81).

IDH

IDH (isocitrate dehydrogenase) is a dimeric protein encoded by the icd gene. Phosphorylation of this enzyme plays a key role in the partition of carbon between the TCA cycle and the glyoxylate shunt. We will discuss this enzyme in the context of the shunt.

2-Ketoglutarate Dehydrogenase

An appreciable amount of our information on E. coli 2-ketoglutarate dehydrogenase comes by analogy with the closely related enzyme pyruvate dehydrogenase, with which it shares the lipoamide dehydrogenase subunit (the lpd gene product) (27, 31a, 32, 61, 71). Lipoic acid is attached to a specific lysine residue close to the the N terminus of the E2 (sucB) subunit by lipoate ligase. This modification is essential for enzyme activity in that this coenzyme acts as a classical swinging arm in moving enzyme intermediates between two active sites. The protein-bound lipoate accepts the decarboxylation product from thiamine pyrophosphate cofactor of the E1 (sucA) subunit and delivers this product to the second active site in the catalytic portion of SucB to form succinyl-CoA. The E3 (lpd) subunit then reoxidizes the E2-bound dihydrolipoate acid moiety. The three subunits (encoded by the sucAB and lpd genes) that make up the active enzyme are found in huge complexes of molecular weight 5 10⁶ to 10 10⁶ that form particles of 30 to 40 nm (significantly larger than ribosomes). The enzyme is too large for direct structural analysis by current methods, and thus parts of the complex have been studied by crystallography and nuclear magnetic resonance with considerable success (61, 71). The enzyme consists of a core of 24 copies of the E2 (sucB) subunit together with 12 copies of each of the other two subunits. However, it seems that this enzyme may not have a strictly defined structure (61), and complexes of various stoichiometries probably exist in vivo. The analogy between 2-ketoglutarate dehydrogenase and pyruvate dehydrogenase is evident not only from the reaction chemistries and deduced amino acid sequences, but also from the common quaternary structure (61, 71).

Phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase are capable of catalyzing a futile cycle which results in the wasteful hydrolysis of ATP (7). Surprisingly, this futile cycle is enhanced in mutants lacking 2-ketoglutarate dehydrogenase (7). This observation suggests that the TCA cycle may provide a protection against excess ATP consumption.

Succinyl-CoA Synthetase

The enzymology of E. coli succinyl-CoA synthetase (more properly called succinate thiokinase) is well studied (66). The enzyme is an α _2β2 tetramer, and the reaction proceeds through a phosphorylated enzyme intermediate in which a specific His residue of the a subunit is modified. The enzyme has been crystallized, and a high-resolution X-ray structure was recently reported that includes the phosphohistidine residue, the first structural determination of this modification in situ (97).

During aerobic growth, succinyl-CoA synthetase generates ATP (GTP is also produced in vitro). This substrate-level phosphorylation serves to use the energetic thioester bond of the succinyl-CoA produced by 2-ketoglutarate dehydrogenase to produce a nucleotide triphosphate. During anaerobic growth the enzyme serves to convert the succinate produced by fumarate reductase to the succinyl-CoA needed for biosynthesis. This point was recently demonstrated by isolation of phage Mu insertions into sucCD (60). These strains fail to grow anaerobically on glucose unless supplemented with lysine, methionine, and δ-aminolevulinic acid. The sucCD genes were originally identified by the similarity of the open reading frames encoded downstream of the sucAB genes to those of the mammalian succinate thiokinase subunits (63), and thus the Mu insertions document the physiological function of these genes.

Succinate Dehydrogenase

Succinate dehydrogenase of E. coli is encoded by four genes in an operon located within the cluster of TCA cycle genes (for review see reference 2). The sdhA gene encodes a flavoprotein subunit containing a covalently bound flavin adenine dinucleotide moiety, whereas the sdhB gene encodes an iron-sulfur protein (32). The sdhC and sdhD genes encode two very hydrophobic membrane proteins that serve to anchor the hydrophilic flavoprotein and iron-sulfur protein subunits to the cytoplasmic membrane and also participate in electron transport (the SdhC protein is cytochrome b ₅₅₆) (32).

The SdhA and SdhB proteins have remarkable sequence similarities to the proteins encoded by the frdA and frdB genes of the anaerobic fumarate reductase, suggesting a common evolutionary origin (32). (The flavoprotein and iron-sulfur protein subunits of fumarate reductase are also anchored to the membrane by a pair of very hydrophobic membrane proteins unrelated to SdhC and SdhD.) Succinate dehydrogenase and fumarate reductase catalyze the same reaction, the interconversion of succinate and fumarate. Indeed, the original cloning of the fumarate reductase operon resulted from attempts to clone the succinate dehydrogenase genes (multiple copies of frdABCD permit sdh mutants to grow aerobically) (30, 32). As discussed above, these two enzyme complexes are differentially expressed. Succinate dehydrogenase is only present in aerobically grown cells, where it functions to donate electrons to the respiratory chain. In contrast, fumarate reductase is present only in anaerobically grown cells, where the enzyme synthesizes succinate in the branched mode of the TCA cycle and also provides a terminal oxidase to aid mixed acid fermentation (and to provide energy for anaerobic growth on nonfermentable carbon sources when fumarate is present in the medium).

Fumarase

E. coli contains three distinct fumarases encoded by the fumA, fumB, and fumC genes (4, 31, 32, 98). The first two enzymes are 90% identical and belong to a novel class of fumarases that are dimeric, oxygen-labile iron-sulfur proteins (32, 98). FumC is a typical oxygen-stable tetrameric fumarase that strongly resembles the mammalian and yeast mitochondrial enzymes. FumA is clearly the enzyme designed for TCA cycle function, whereas FumB is derepressed under anaerobic conditions and functions as a malate dehydratase in the branched TCA cycle and also converts malate to fumarate to be utilized as an anaerobic electron acceptor (98). FumC fumarase is encoded by a gene adjacent to fumA, but the two genes are regulated differently. fumA is regulated in the same manner as the other TCA cycle genes, whereas fumC expression does not respond to O₂ or glucose levels. However, fumC has been reported to be regulated by the SoxRS regulon, which functions to protect redox balance and metabolism from oxidative stress (56). Replacement of the oxidation-labile FumA with higher levels of the stable FumC would serve such a protective function. However, several other TCA cycle enzymes are labile iron-sulfur proteins, and thus increased FumC activity alone seems unlikely to spare the TCA cycle from oxidative stress. FumC has recently been crystallized (93).

Malate Dehydrogenase

The malate dehydrogenase of E. coli is a dimer of the protein encoded by the mdh gene, and the deduced amino acid sequence is closely similar to that of mammalian mitochondrial enzymes and distinct from the mammalian cytosolic enzymes (88, 90; for review see reference 62). Crystal structures of the enzyme in the presence and absence of citrate have recently been reported (33). Mutations in a locus distinct from mdh also result in a specific loss of malate dehydrogenase activity (12). These mutant strains produce activity when transformed with multicopy mdh plasmids (90), and thus this second locus has been assigned a regulatory role. These mutants should be further explored. The putative anaerobic role for the mdh gene product has not been demonstrated, and it seems possible that the anaerobic conversion of oxaloacetate to malate could be catalyzed by consecutive action of aspartate transaminase and aspartase (32). The latter enzyme is known to be derepressed under anaerobic conditions (98).

TCA CYCLE MYSTERIES

There are several unexpected aspects of the TCA cycle that remain unexplained. First, icd mutants are readily isolated by selection for strains resistant to the DNA gyrase inhibitor nalidixic acid (34, 47). Second, gltA icd double mutants have been reported to contain citrate (perhaps this is due to minimal function of the cryptic enzyme discussed above) (48).

Another mystery is that the succinate requirement for aerobic growth of sucA or lpd mutants on glucose is suppressed by mutations within the sdh operon (14, 15). It therefore seems that E. coli has another source of succinate, and utilization of this supply for biosynthesis competes with consumption by succinate dehydrogenase. This suppression phenomenon was observed with lpd deletion mutants (14), and thus the caveat of residual α-ketoglutarate dehydrogenase function does not seem germane. On the other hand, biochemical evidence for a lipoamide dehydrogenase clearly distinct from Lpd has been reported (77). However, the purified enzyme failed to replace Lpd in supporting α-ketoglutarate dehydrogenase activity measured in vitro (77). It seems probable that E. coli requires only small amounts of succinate, since succinyl-CoA is used only catalystically in amino acid biosynthesis. It also seems that E. coli has a source of succinyl-CoA other than succinyl-CoA synthetase since sucCD mutants grow anaerobically on fucose (but not on glucose) and second-site revertants of sucCD mutations that restore aerobic growth on acetate have been isolated (60). It seems possible that these mutations might activate the anaerobic pathway used on fucose.

REGULATION OF EXPRESSION OF THE GENES OF THE TCA CYCLE

The levels of the TCA cycle enzymes are regulated at the transcriptional level by interaction of two global regulatory systems, catabolite (glucose) repression and the ArcAB two-component system (29, 31, 32, 55, 63, 80). Catabolite repression of the TCA cycle enzymes seems to follow the typical pattern in that transcription is decreased in crp mutants lacking the Crp transcriptional activator (63, 83). However, it should be noted that several of the known TCA cycle promoters have no readily apparent Crp DNA-binding sequence (63, 83). The ArcAB system was discovered by the release of the anaerobic repression of the sdhCDAB operon (42, 43, 55). It was subsequently demonstrated that this system regulates the effects of oxygen availability on the levels of the TCA cycle enzymes. We shall defer to the sections of this volume that deal with control of gene expression by Crp (chapter 85) and ArcAB (chapter 95). The derepression of fumarate reductase, fumarase B, and aspartase is due to regulation by FNR (chapter 95).

An exception to simple ArcAB/Crp regulation is the transcription of the lpd gene that encodes the E3 (dihydrolipoamide dehydrogenase) subunit of both the 2-ketogluturate and pyruvate dehydrogenases. lpd has recently been shown to be transcribed from either of two promoters, the pdh promoter located upstream of the pdhR reguatory gene and the lpd promoter (74). Transcription initiating at the pdh promoter proceeds through pdhR and aceEF before lpd is transcribed. PdhR is a negative regulator of the pdh promoter, and thus the synthesis of E3 is increased by the presence of pyruvate. The lpd promoter is also regulated by the ArcAB and Crp systems, which have little effect on the pdh promoter (74).

THE GLYOXYLATE BYPASS

The glyoxylate bypass (see Fig. 1) is essential for growth on carbon sources such as acetate or fatty acids because this pathway allows the net conversion of acetyl-CoA to metabolic intermediates. Strains lacking this pathway fail to grow on these carbon sources since acetate carbon entering the TCA cycle is quantitatively lost as CO₂ (45) and thus there is no means to replenish the dicarboxylic acids consumed in amino acid biosynthesis. The pathway involves the synthesis of three enzymes. Two of these, isocitrate lyase and malate synthase A, act to convert some of the TCA cycle carbon from isocitrate to malate. Isocitrate lyase competes with the TCA cycle enzyme, IDH (isocitrate dehydrogenase), for isocitrate, and the third enzyme, IDH kinase/phosphatase, is needed to decrease the activity of IDH to allow isocitrate lyase to effectively compete for isocitrate.

THE ENZYMES OF THE GLYOXYLATE BYPASS

The two enzymes that interconvert the intermediates of the glyoxylate bypass are isocitrate lyase and malate synthase A, encoded by the aceA and aceB genes, respectively. The AceB protein is called malate synthase A (A for induction by acetate) to distinguish it from a second malate synthase, malate synthase G (G for induction by glycolate), encoded by the glcB gene, which functions in growth on glycolate or glyoxylate as sole carbon sources (chapter 21). The two malate synthases are readily distinguished by differing stabilities and inhibitor patterns (68). The enzymology of malate synthase A seems little explored, although the deduced amino acid sequence is known (Table 1). The enzyme does not appear to have been purified to homogeneity, and the subunit structure has not been reported. In contrast, isocitrate lyase, a tetrameric protein, has been purified, and the active site and mechanism have been studied in some detail (19, 20). The enzyme has been crystallized (1), but no structure is yet available. Active isocitrate lyase is phosphorylated on a histidine residue, and this modification seems essential for activity. However, the identity of the histidine residue and the role of the modification are unclear (20). The third protein, the IDH kinase/phosphatase, provides the most novel aspect of the glyoxylate bypass and will be discussed in detail later in this chapter.

REGULATION OF IDH AND THE GLYOXYLATE BYPASS

In E. coli and S. typhimurium, IDH is regulated by phosphorylation. The function of this phosphorylation cycle is to control the flow of isocitrate through the glyoxylate bypass (Fig. 1). During growth on acetate, about 75% of the IDH is converted to the inactive phosphorylated form. Inhibition of IDH slows the TCA cycle and thus forces isocitrate through the bypass (6, 54).

The first insight into the regulation of IDH came in the early 1970s when Bennett and Holms (5, 36) reported that the activity of this enzyme was increased when E. coli made the transition between growth on acetate and growth on preferred carbon sources such as glucose or pyruvate. Increased activity did not simply result from induction of IDH expression since it occurred even in the presence of protein synthesis inhibitors (5, 36). Lowry and coworkers (57) came to similar conclusions but by quite a different route. They found that addition of glucose to a culture growing on acetate produced a metabolic crossover at IDH; the cellular level of isocitrate went down while that of 2-ketoglutarate increased. This metabolic crossover suggested that IDH had been activated during this transition (57). However, the mechanism responsible for regulation of IDH was not reported until 1979, when it was demonstrated that IDH activity was controlled by phosphorylation (24, 25).

Regulation of E. coli IDH by phosphorylation was a surprising result. Although protein phosphorylation had been known for many years in eukaryotes (11, 21, 37), conventional wisdom held that bacteria did not use this regulatory strategy. The lone exception appeared to be bacteriophage T7, a bacterial virus which encodes a protein kinase activity capable of phosphorylating RNA polymerase during the infection cycle (75, 79). The discovery that IDH is regulated by phosphorylation, and the simultaneous identification of a variety of other protein kinase activities in extracts of E. coli and S. typhimurium, demonstrated that protein phosphorylation was, in fact, a universal regulatory strategy (1, 13, 24, 92).

Structural Basis of the Inhibition of IDH

Phosphorylation of IDH results in total inactivation of the enzyme (6, 25, 51). The extent of this inhibition is a striking contrast to that in most phosphorylated enzymes, which undergo more subtle changes in their properties. IDH is inactivated because the phosphorylated serine lies within the active site (78). In the active dephosphorylated enzyme, this serine forms a hydrogen bond with isocitrate, one of the substrates. Phosphorylation of IDH blocks isocitrate binding by disrupting this hydrogen bond and by introducing electrostatic repulsion between this phosphate and isocitrate (17, 18, 38, 39, 40).

Enzymology of the IDH Phosphorylation Cycle

The enzymes which phosphorylate and dephosphorylate IDH exhibit a number of peculiar features. IDH kinase and IDH phosphatase are encoded by the same gene, aceK, and reside on the same polypeptide (44, 49, 50). This phosphatase activity has an absolute requirement for ATP or ADP, but is not supported by nonhydrolyzable ATP analogs (50; unpublished observations). IDH kinase/phosphatase also catalyzes a third activity, an ATPase which is more active than either IDH kinase or IDH phosphatase (84). IDH kinase/phosphatase appears to catalyze all three activities at the same active site. This model was first suggested by the observation that a variety of mutations have parallel effects on these activities. For example, one class of mutant proteins possess kinase, phosphatase, and ATPase activities which exhibit drastic reductions in affinity of phospho-IDH. Furthermore, all three activities of one of these proteins have reduced affinity for ATP (unpublished observations). Finally, a mutation in a conserved ATP-binding site motif eliminates both IDH kinase and IDH phosphatase activities (86). Affinity labeling of IDH kinase/phosphatase with an ATP analog also supports a single active site on IDH kinase/phosphatase (89). This analog yielded parallel inhibition of IDH kinase and IDH phosphatase yet, in a preliminary experiment, appeared to label a single peptide.

A likely model for the mechanism of this protein proposes that the IDH kinase and IDH phosphatase reactions occur in the same active site and that the phosphatase reaction results from the back reaction of IDH kinase tightly coupled to ATP hydrolysis. According to this model, the phosphatase reaction requires the formation of a ternary complex between IDH kinase/phosphatase, ADP, and phospho-IDH. The phosphate group of phospho-IDH is then transferred to ADP (the kinase back reaction) and then to water (an ATPase reaction). This sequence of steps results in the net dephosphorylation of phospho-IDH (the phosphatase reaction) (52, 84).

Regulation of the Glyoxylate Bypass by IDH Phosphorylation

One role of the IDH phosphorylation cycle is to regulate the branch point between the glyoxylate bypass and the TCA cycle during steady-state growth on acetate or fatty acids (see above). Phosphorylation of IDH diverts some of the flux from the TCA cycle to the glyoxylate bypass. The immediate effect of phosphorylation is to inhibit IDH activity. The resulting increase in the level of isocitrate increases the velocity of isocitrate lyase, the first enzyme of the bypass. Mathematical analyses have demonstrated that saturation of IDH with isocitrate (which occurs during growth on acetate) makes it impossible to directly regulate isocitrate lyase activity. Regulation can only be achieved indirectly through control of IDH activity (54). Mutant strains which are deficient in IDH kinase failed to grow on acetate, suggesting that the phosphorylation of IDH is required for use of the glyoxylate bypass (53).

IDH kinase/phosphatase also controls the glyoxylate bypass during transitions between carbon sources. For example, addition of a preferred carbon source (e.g., glucose or pyruvate) to a culture growing on acetate renders the glyoxylate bypass unnecessary. Under these conditions, the cell shuts this pathway down by dephosphorylating IDH. Inhibition of the bypass results because the activation of IDH draws isocitrate through the TCA cycle. The resulting decrease in isocitrate concentration yields a proportional decrease in the velocity of isocitrate lyase (54).

What Controls the IDH Phosphorylation Cycle?

A variety of metabolites have been identified which affect IDH kinase/phosphatase in vitro. These metabolites activate IDH phosphatase and inhibit IDH kinase (51, 65). During growth on acetate, it appears that isocitrate and 3-phosphoglycerate participate in the control of the IDH phosphorylation cycle. These effectors probably act as general indicators of the levels of metabolic intermediates and thus of the need for isocitrate to be directed to the glyoxylate bypass. For example, depletion of these metabolites would result in increased phosphorylation of IDH, forcing more isocitrate through the bypass. However, isocitrate and 3-phosphoglycerate are not responsible for the dephosphorylation of IDH which results from the addition of preferred carbon sources such as glucose, since their levels fall under these conditions. Dephosphorylation of IDH during these metabolic transitions is probably promoted, at least in part, by pyruvate, since the level of this metabolite rises dramatically upon addition of glucose (22, 57).

Although the glyoxylate bypass can provide metabolic intermediates, the TCA cycle is more efficient in providing energy. The cell must, therefore, precisely balance the flux of isocitrate between these competing pathways during growth on acetate. The energy requirements of the cell appear to be monitored through AMP levels. Like the other metabolites discussed above, AMP activates IDH phosphatase and inhibits IDH kinase. AMP is a particularly attractive choice for monitoring the energy needs of the cell because, if the adenylate kinase reaction is at equilibrium, the level of AMP will vary as the square of ADP concentration. An increase in AMP, signaling a depletion of cellular energy, would yield a net dephosphorylation of IDH, diverting more isocitrate through the TCA cycle, whereas a surplus of energy, indicated by a low level of AMP, would have the opposite effect. An open question is the regulation of IDH activity during anaerobic growth when the TCA cycle functions in the branched mode.

Sensitivity Amplification and the Glyoxylate Bypass

The regulation of the glyoxylate bypass appears to be exquisitely sensitive to the metabolic state of the cell. This conclusion is supported by a variety of theoretical analyses and by experiments performed in vitro. A high degree of sensitivity amplification is achieved by simultaneous activation of IDH phosphatase and inhibition of IDH kinase by its effectors. This type of sensitivity amplification is termed a multistep effect. The IDH phosphorylation cycle also appears to be subject to zero-order ultrasensitivity, a form of sensitivity amplification which occurs in covalent regulatory systems when the concentration of the interconvertible protein (in this case IDH) exceeds the Michaelis constants of the converter enzymes (26, 51). A third mechanism of sensitivity enhancement, termed a branch point effect, results from the profound difference in the affinities of IDH and isocitrate lyase for isocitrate (Michaelis constants of 8 and 600 mM, respectively) (54). As a result of the branch point effect, the flux of the glyoxylate bypass is strikingly sensitive to the phosphorylation state of IDH. These three mechanisms for sensitivity amplification combine to produce a system in which very subtle changes in metabolic signals have the potential for producing profound changes in the flux through the glyoxylate bypass.

In addition to responding to changes in the external environment, the IDH phosphorylation cycle must be capable of adapting to the conditions which prevail inside the cell. For example, the cellular level of IDH can vary by at least a factor of 2 between different strains of E. coli. The effect of this difference in IDH levels would be amplified by the branch point effect (see above), potentially preventing growth on acetate. However, the IDH phosphorylation cycle responds to these differences by altering the fractional phosphorylation of IDH so that a constant level of IDH activity is maintained under these growth conditions. This cycle could even compensate for a 15-fold overproduction of IDH, a condition which clearly has the potential for pathological consequences (53). It seems likely that this response to the cellular level of IDH resulted because the control of IDH kinase/phosphatase by a variety of metabolites provides a particularly effective feedback control mechanism.

Is precise control of IDH phosphorylation necessary? The tolerance of E. coli to variation in IDH activity was tested using mutant strains which had defects in IDH kinase/phosphatase. The effect of excess IDH activity was determined by comparison of strains with null mutations in aceK. A twofold increase in IDH activity reduced the growth rate on acetate while a fourfold increase yielded nearly complete inhibition of growth (53). The minimum level of IDH required for growth on acetate was determined by coexpression of wild-type aceK with an allele of aceK whose product selectively lacked IDH phosphatase activity. The cells tolerated a 50% reduction in IDH activity without apparent effect on growth rate. However, further reductions in IDH activity inhibited growth, with arrest occurring when this level dropped to 15% of the wild type (41). Thus, the striking precision exhibited by the IDH phosphorylation cycle in vivo is not absolutely essential. However, in nature, even small differences in growth rate can yield dramatic differences during competition between organisms for limiting resources.

Balancing Environmental Sensitivity and Futile Cycling

A regulatory system which employs covalent control must balance the benefits of a rapid response to a signal with the need to minimize the loss of cellular energy which results from the cyclic modification of the target protein (i.e., futile cycling). Insight into the minimum level of IDH kinase/phosphatase required for growth on acetate came from an unexpected source. Although mutation of the consensus ATP-binding site reduced the IDH kinase and IDH phosphatase activities by factors of at least 100 both in vivo and in vitro, the altered protein retained sufficient kinase activity to support growth on acetate. It thus appears that wild-type IDH kinase/phosphatase is maintained in massive excess over the level required for steady-state phosphorylation of IDH (84). A likely function for this excess IDH kinase/phosphatase would be to allow rapid responses to changes in the available carbon source. For example, when pyruvate is added to cultures growing on acetate, the cells dephosphorylate IDH, increasing its activity, thus inhibiting the flux of isocitrate through the glyoxylate bypass (see above). The ability to control the expression of wild-type IDH kinase/phosphatase by using a clone of a wild-type aceK gene provided a method for determining the effect of the level of this protein on the response rate. The rate of the pyruvate-induced dephosphorylation of IDH was proportional to the level of IDH kinase/phosphatase, demonstrating that IDH kinase/phosphatase represented the primary rate-limiting step in this metabolic transition (85).

GENETIC CONTROL OF THE GLYOXYLATE BYPASS

The genes which encode the metabolic and regulatory enzymes of the glyoxylate bypass reside in the same operon, aceBAK (9, 53, 59) (Fig. 3). The metabolic enzymes, malate synthase and isocitrate lyase, are encoded by aceB and aceA, whereas aceK encodes IDH kinase/phosphatase. The operon is expressed from a single promoter during growth on acetate (9). Expression is induced during growth on acetate or fatty acids, but induction can be prevented by the presence of preferred carbon sources (e.g., glucose, glycerol, or pyruvate) (45). Expression of the glyoxylate bypass operon also responds to aerobic respiration, with repression occurring under anaerobic conditions (42, 43).

Fig. 3The glyoxylate bypass operon. Malate synthase, isocitrate lyase, and IDH kinase/phosphatase are encoded by aceB, aceA, and aceK. These genes are transcribed from a single promoter

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The expression of aceBAK is affected by IHF, a histonelike protein which is abundant in E. coli. IHF binds to two sites which are upstream of the promoter. Binding of IHF to the promoter-proximal site yields a fivefold increase in transcriptional activity (E. Resnik, N. Ramani, M. Freundlich, and D. C. LaPorte, manuscript in preparation). The aceBAK operon is regulated, at least in part, by a repressor protein encoded by iclR (46, 59, 64, 87). The IclR protein binds to a site that overlaps the –35 region of the aceBAK promoter (9, 64). Release of this repressor upon adaptation to growth on acetate or fatty acids is presumably responsible for induction of operon expression. By analogy with other systems, this release presumably involves binding of some metabolic intermediate. Acetate can be ruled out since fatty acids also induce expression of aceBAK (67). However, the metabolic intermediate cannot yet be confidently identified.

The response of aceBAK to fatty acids is mediated, in part, by FadR (58, 59). FadR was initially identified because it represses the genes encoding the enzymes of fatty acid degradation (67). FadR was subsequently shown to activate the transcription of fabA, a gene whose product participates in unsaturated fatty acid biosynthesis, and DNA binding by FadR is specifically antagonized by CoA esters of long-chain fatty acids (35). Mutations in fadR result in increased expression of aceBAK on repressing media, although the effects of these mutations are much smaller than those observed for iclR (59).

Expression of the glyoxylate bypass operon, like TCA cycle gene expression, responds to aerobic respiration, with repression occurring under anaerobic conditions (42, 43), and the regulation is also mediated by the ArcAB global regulatory system (chapter 95). The expression of the glyoxylate bypass operon may also be controlled by the product of fruR, a global regulatory protein first identified by its effects on fructose metabolism (8). In vitro, the FruR protein binds to a site upstream of the aceBAK promoter (76). However, the physiological significance of this site remains uncertain since deletion of this region has only a slight effect on aceBAK expression (Resnik et al., submitted).

Polarity in aceBAK

Although the genes of the aceBAK operon are expressed from the same promoter (Fig. 3), the relative cellular levels of malate synthase:isocitrate lyase:IDH kinase/phosphatase are approximately 0.3:1:0.003. The upshift in expression between aceB and aceA results from differences in translational efficiency. In contrast, inefficient translation and premature transcriptional termination contributed to the downshift in expression between aceA and aceK. Premature transcriptional termination occurs within aceK and appears to result from inefficient translation. The sequences responsible for inefficient expression of aceK lie within the ribosome-binding site of this gene, a surprising result since this site contains a good match to the Shine-Dalgarno ribosome-binding sequence (10).

FUTURE DIRECTIONS

Although significant gaps remain in our knowledge of the carbon flow of the TCA cycle and glyoxylate bypass, future work in this area seems likely to be targeted at the detailed enzymology at the three-dimensional structural level. Understanding of the mechanisms that regulate gene expression in these pathways is also an area that should provide rewarding insights into basic cell physiology.

ACKNOWLEDGMENT

We thank John R. Guest for his valuable comments on a prior draft of this review.