Biosynthesis of o-succinylbenzoic acid. II: Properties of o-succinylbenzoic acid synthase, an enzyme involved in vitamin K2 biosynthesis

The enzyme system (OSB2 synthase) catalyzing the synthesis of o-succinylbenzoic acid from isochorismic acid and alpha-ketoglutaric acid in the presence of thiamine pyrophosphate was isolated from Escherichia coli AN 154 and characterized. The purification factor of the enzyme did not increase during column chromatography on Sephadex G-200 or chromatofocusing, suggesting that the OSB synthase is labile. Chromatography on DEAE-Sephadex A-50 or A-25 showed that an enzyme activity separated from fractions containing OSB synthase that decarboxylates alpha-ketoglutarate. This activity is provisionally referred to as the decarboxylating "subunit" or decarboxylating activity of OSB synthase. Both the "subunit" and the holoenzyme were characterized with respect to pH optimum, temperature optimum, and KM values. The OSB synthase loses all activity during treatment with EDTA and activity is most efficiently restored with Mn2+. The activity of the decarboxylating subunit did not depend on Mn2+. When the decarboxylating fraction was incubated with alpha-ketoglutarate and thiamine pyrophosphate, succinic semialdehyde could be isolated as its hydrazone. After treatment of the incubation mixture with phosphorylase a compound was isolated which is most likely the thiamine adduct of succinic semialdehyde.     

Biosynthesis of o-succinylbenzoic acid in Bacillus subtilis: identification of menD mutants and evidence against the involvement of the alpha-ketoglutarate dehydrogenase complex.

The biosynthesis of o-succinylbenzoic acid (OSB), the first aromatic intermediate involved in the biosynthesis of menaquinone (vitamin K2) is demonstrated for the first time in the gram-positive bacterium Bacillus subtilis. Cell extracts were found to contain isochorismate synthase, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) synthase-alpha-ketoglutarate decarboxylase and o-succinylbenzoic acid synthase activities. An odhA mutant which lacks the decarboxylase component (usually termed E1, EC 1.2.4.2, oxoglutarate dehydrogenase [lipoamide]) of the alpha-ketoglutarate dehydrogenase complex was found to synthesize SHCHC and form succinic semialdehyde-thiamine pyrophosphate. Thus, the presence of an alternate alpha-ketoglutarate decarboxylase activity specifically involved in menaquinone biosynthesis is established for B. subtilis. A number of OSB-requiring mutants were also assayed for the presence of the various enzymes involved in the biosynthesis of OSB. All mutants were found to lack only the SHCHC synthase activity.     

Thiamine pyrophosphate requirement for o-succinylbenzoic acid synthesis in Escherichia coli and evidence for an intermediate.

Cell-free extracts of various strains of Escherichia coli synthesize the menaquinone biosynthetic intermediate o-succinylbenzoic acid (OSB) when supplied with chorismic acid, 2-ketoglutaric acid, and thiamine pyrophosphate (TPP). To assay for OSB synthesis, 2-[U-14C]ketoglutaric acid was used as substrate, and the synthesized OSB was examined by radiogas chromatography (as the dimethyl ester). [U-14C]Shikimic acid also gave rise to radioactive OSB if the cofactors necessary for enzymatic conversion to chorismic acid were added. Use of 2-[1-14C]ketoglutaric acid does not give rise to labeled OSB. In the absence of TPP during the incubations, OSB synthesis was much reduced; these observations are consistent with the proposed role for the succinic semialdehyde-TPP anion as the reagent adding to chorismic acid. Extracts of cells from menC and menD mutants did not form OSB separately, but did so in combination. There was evidence for formation of a product, X, by extracts of a menC mutant incubated with chorismic acid, TPP, and 2-ketoglutaric acid; X was converted to OSB by extracts of a menD mutant. It appears that the intermediate, X, is formed by one gene product and converted to OSB by the second gene product.     

Role of the entC gene in enterobactin and menaquinone biosynthesis in Escherichia coli

Tn10 mutants of Escherichia coli MC4100 were screened for their inability to grow under iron deficiency and for their inability to grow under anaerobiosis in the presence of fumarate as an electron acceptor. A strain so obtained (E. coli PBB1) lacked the ability to convert chorismic acid to isochorismic acid. This shows that the gene (entC) encoding isochorismate synthase was mutated. E. coli PBB1 did not produce any detectable amounts of menaquinones (vitamin K2) or enterobactin. When supplemented with isochorismic acid this strain produced menaquinones, indicating that isochorismic acid is involved not only in enterobactin but also in menaquinone biosynthesis. The entC gene was isolated and was shown to be part of the enterobactin gene cluster: It was located on a DNA fragment (9 kb in length) which also carried the entA gene. The DNA fragment was identified by restriction site mapping and was compared to a previously published map of the enterobactin gene cluster. The entC gene on this fragment responds not only to conditions (iron deficiency) that stimulate enterobactin biosynthesis but also to anaerobiosis which results in increased isochorismic acid formation and increased menaquinone biosynthesis. We conclude that isochorismic acid, isochorismic synthase, and the gene (entC) encoding this enzyme are involved in catalytic events at a metabolic branch point from which both enterobactin and menaquinones originate.     

Vitamin K (menaquinone) biosynthesis in bacteria: high-performance liquid chromatographic assay of the overall synthesis of o-succinylbenzoic acid and of 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid synthase

An early enzyme in menaquinone (vitamin K2) biosynthesis is the synthase forming 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) from isochorismic acid. In turn, SHCHC is aromatized to o-succinylbenzoic acid (OSB) by OSB synthase. An assay for the combined activity of these two enzymes ("overall OSB synthesis") has been developed using a high-performance liquid chromatographic method for the quantitation of OSB. The assay, which measures as little as 0.1 nmol of OSB, is vastly superior to the radiogas chromatographic method previously used to estimate overall OSB synthesis. To measure SHCHC synthase activity separately, the enzymatically formed SHCHC is converted nonenzymatically to OSB (heating to 80 degrees C, pH 10, 10 min), which is then quantitated by the HPLC assay. The preparation of the substrate, isochorismic acid, and its purification by preparative HPLC are also described.     

Isochorismate hydroxymutase from Rubiaceae cell suspension cultures

The enzyme catalysing the isomerisation of chorismic to isochorismic acid (isochorismate hydroxymutase E.C. 5.4.99.6) has been detected in protein preparations of various cell suspension cultures derived from plants of Rubiaceae species.Abbreviations OSB o-Succinylbenzoic acid - Tris Tris(hydroxymethyl)aminomethane - DEAE Diethylaminoethyl cellulose     

Clustering of isochorismate synthase genes menF and entC and channeling of isochorismate in Escherichia coli.

There are two isochorismate synthase genes entC and menF in Escherichia coli. They encode enzymes (isochorismate synthase, EC 5.4.99.6) which reversibly synthesize isochorismic acid from chorismic acid. The genes share a 24.2% identity but are differently regulated. Activity of the MenF isochorismate synthase is significantly increased under anaerobic conditions whereas the activity of the EntC isochorismate synthase is greatly stimulated during growth in an iron deficient medium. Isochorismic acid synthesized by EntC is mainly channeled into enterobactin synthesis whereas isochorismic acid synthesized by MenF is mainly channeled into menaquinone synthesis. When menF or entC were separately placed onto overexpression plasmids and the plasmids introduced into a menF(-)/entC(-) double mutant in two separate experiments, the isochorismate formed was fed into both, the menaquinone and the enterobactin pathway. Moreover, in spite of a high isochorismate synthase activity menaquinone and enterobactin formation were not fully restored, indicating that isochorismate was lost by diffusion. Thus, under these conditions channeling was not observed. We conclude that in E. coli the chromosomal position of both menF and entC in their respective clusters is a prerequisite for channeling of isochorismate in both pathways.     

Origin of p-aminobenzoic acid from chorismic rather than iso-chorismic acid in Enterobacter aerogenes and Streptomyces species

Enzyme extracts from Enterobacter aerogenes (62-1), Streptomyces aminophilus, and Streptomyces coelicolor were used to investigate the biosynthesis of p-aminobenzoic acid. The enzyme preparations from E. aerogenes and S. aminophilus contained both p-aminobenzoate synthase and iso-chorismate synthase activity, and were able to convert both chorismic and iso-chorismic acid to p-aminobenzoic acid. The apparent KM for chorismic acid was, however, significantly lower than that for iso-chorismic acid, while the Vmax was identical for both substrates in both enzyme systems. The enzyme preparations from S. coelicolor did not contain iso-chorismate synthase activity and p-aminobenzoic acid synthesis took place in this system from chorismic acid only. It is concluded that iso-chorismic acid is not an obligatory intermediate in p-aminobenzoic acid biosynthesis in these organisms.     

Characterization and regulation of p-aminobenzoic acid synthase from Streptomyces griseus

p-Aminobenzoic acid synthase (PABA synthase) of Streptomyces griseus catalyses the conversion of chorismic acid to p-aminobenzoic acid (PABA), a precursor of the aromatic p-aminoacetophenone moiety of candicidin, a polyene macrolide antibiotic. This enzyme uses glutamine or ammonia as amino donors for PABA formation. Enzyme extracts converted [14C]chorismic acid to labelled PABA. PABA synthase was present in S. griseus IMRU 3570 only during the antibiotic producing phase. No detectable levels of the enzyme were found in cell-free extracts of nonproducing mutants of S. griseus obtained after UV mutagenesis. PABA synthase activity was found also in Streptomyces coelicolor var. aminophilus, producer of the polyene macrolide antibiotic fungimycin, but it was not present in extracts of several other streptomycetes that do not produce aromatic polyene macrolide antibiotics. PABA synthase (amidotransferase) activity was partially purified by DEAE-Bio-gel and Sephacryl S-200 filtrations. The estimated molecular weight was 50000. PABA synthase was repressed by aromatic amino acids and PABA but not by anthranilic acid. Inorganic phosphate strongly repressed but did not inhibit PABA synthase activity.     

Identification of Bacillus subtilis men mutants which lack O-succinylbenzoyl-coenzyme A synthetase and dihydroxynaphthoate synthase

Menaquinone (vitamin K2)-deficient mutants of Bacillus subtilis, whose growth requirement is satisfied by 1,4-dihydroxy-2-naphthoic acid but not by o-succinylbenzoic acid (OSB), have been analyzed for enzymatic defects. Complementation analysis of cell-free extracts of the mutants revealed that there are two groups, as already indicated by genetic analysis. The missing enzyme in each group was identified by complementation of the cell-free extracts with o-succinylbenzoyl-coenzyme A (CoA) synthetase and dihydroxynaphthoate synthase extracted from Mycobacterium phlei. Mutants found to lack dihydroxynaphthoate synthase, and which therefore complement with dihydroxynaphthoate synthase of M. phlei, were designated as menB; those lacking o-succinylbenzoyl-CoA synthetase, and therefore complementing with o-succinylbenzoyl-CoA synthetase, were designated as menE. The menB mutants RB413 (men-325) and RB415 (men-329), when incubated with [2,3-14C2]OSB, produced only the spirodilactone form of OSB in a reaction that was CoA and adenosine 5'-triphosphate dependent.     

Evolution of enzymatic activity in the enolase superfamily: structural and mutagenic studies of the mechanism of the reaction catalyzed by o-succinylbenzoate synthase from Escherichia coli

o-Succinylbenzoate synthase (OSBS) from Escherichia coli, a member of the enolase superfamily, catalyzes an exergonic dehydration reaction in the menaquinone biosynthetic pathway in which 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) is converted to 4-(2'-carboxyphenyl)-4-oxobutyrate (o-succinylbenzoate or OSB). Our previous structural studies of the Mg(2+).OSB complex established that OSBS is a member of the muconate lactonizing enzyme subgroup of the superfamily: the essential Mg(2+) is coordinated to carboxylate ligands at the ends of the third, fourth, and fifth beta-strands of the (beta/alpha)(7)beta-barrel catalytic domain, and the OSB product is located between the Lys 133 at the end of the second beta-strand and the Lys 235 at the end of the sixth beta-strand [Thompson, T. B., Garrett, J. B., Taylor, E. A, Meganathan, R., Gerlt, J. A., and Rayment, I. (2000) Biochemistry 39, 10662-76]. Both Lys 133 and Lys 235 were separately replaced with Ala, Ser, and Arg residues; all six mutants displayed no detectable catalytic activity. The structure of the Mg(2+).SHCHC complex of the K133R mutant has been solved at 1.62 A resolution by molecular replacement starting from the structure of the Mg(2+).OSB complex. This establishes the absolute configuration of SHCHC: the C1-carboxylate and the C6-OH leaving group are in a trans orientation, requiring that the dehydration proceed via a syn stereochemical course. The side chain of Arg 133 is pointed out of the active site so that it cannot function as a general base, whereas in the wild-type enzyme complexed with Mg(2+).OSB, the side chain of Lys 133 is appropriately positioned to function as the only acid/base catalyst in the syn dehydration. The epsilon-ammonium group of Lys 235 forms a cation-pi interaction with the cyclohexadienyl moiety of SHCHC, suggesting that Lys 235 also stabilizes the enediolate anion intermediate in the syn dehydration via a similar interaction.     

Subcloning of the enterobactin biosynthetic gene entB: expression, purification, characterization, and substrate specificity of isochorismatase

The Escherichia coli entB gene, coding for the enterobactin biosynthetic enzyme isochorismatase, has been subcloned into the multicopy plasmid pKK223-3 under the control of the tac promoter. The resulting recombinant plasmid pFR1 expresses isochorismatase amounting to over 50% of the total cellular protein. The enzyme has been purified to homogeneity and a convenient assay developed. The enzyme has a Km for isochorismate of 14.7 microM and a turnover number of 600 min-1. By use of 1H NMR spectroscopy, the progress of the reaction was followed with the expected formation of 2,3-dihydro-2,3-dihydroxybenzoate product. Several substrate analogues were also utilized by the enzyme including chorismic acid, the immediate precursor to isochorismic acid in the enterobactin biosynthetic pathway.     

In vitro mutagenesis of Escherichia coli citrate synthase to clarify the locations of ligand binding sites

In vitro mutagenesis techniques have been used to investigate two structure-function questions relating to the allosteric citrate synthase of Escherichia coli. The first question concerns the binding site of alpha-keto-glutarate, which is a structural analogue of the substrate oxaloacetate and yet has been suggested to be an allosteric inhibitor of the enzyme. Using oligonucleotide-directed mutagenesis of the cloned E. coli citrate synthase gene, we prepared missense mutants, designated CS226H----Q and CS229H----Q, in which histidine residues at positions 226 and 229, respectively, were replaced by glutamine. In the homologous pig heart citrate synthase it is known (Wiegand, G., and Remington, S. J. (1986) Annu. Rev. Biophys. Biophys. Chem. 15, 97-117) that the equivalent of His-229 helps to bind oxaloacetate, while the equivalent of His-226 is nearby. Kinetic and ligand binding measurements showed that CS226H----Q had a reduced affinity for oxaloacetate and alpha-ketoglutarate, while CS229H----Q bound oxaloacetate even less effectively, and was not inhibited by alpha-ketoglutarate at all under our conditions. This parallel loss of binding affinities for oxaloacetate and alpha-ketoglutarate, in two mutants altered in residues at the active site of E. coli citrate synthase, strongly suggests that inhibition of this enzyme by alpha-ketoglutarate is not allosteric but occurs by competitive inhibition at the active site. The second question investigated was whether the known inhibition by acetyl-CoA of binding of NADH, an allosteric inhibitor of E. coli citrate synthase, occurs heterotropically, as an indirect result of acetyl-CoA binding at the active site, or directly, by competition at the allosteric NADH binding site. Using existing restriction sites in the cloned E. coli citrate synthase gene, we prepared a deletion mutant which lacked 24 amino acids near what is predicted to the acetyl-CoA-binding portion of the active site. The mutant protein was inactive, and acetyl-CoA did not bind to the active site but still inhibited NADH binding. Thus acetyl-CoA can interact with both the allosteric and the active sites of this enzyme.     

Enhanced acetohydroxy acid synthase III activity in an ilvH mutant of Escherichia coli K-12.

The acetohydroxy acid synthase III isozyme, which catalyzes the first common step in the biosynthesis of isoleucine, leucine, and valine in Escherichia coli K-12, is composed of two subunits, the ilvI and ilvH gene products. A missense mutation in ilvH (ilvH612), which reduced the sensitivity of the enzyme to the end product inhibition by valine, also increased its specific activity and lowered the Km for alpha-acetolactate synthesis. The mutation increased the sensitivity of acetohydroxy acid synthase III to dialysis and heat treatment and reduced the requirement for thiamine pyrophosphate addition to the assay mixture for activity. A strain carrying the ilvH612 mutation grew better than a homologous ilvH+ strain in the presence of leucine. The data indicate that this is a consequence of a more active acetohydroxy acid synthase III isozyme rather than the result of an alteration of the leucine-mediated repression of the ilvIH operon.     

Menaquinone (vitamin K2) biosynthesis in Escherichia coli: synthesis of o-succinylbenzoate does not require the decarboxylase activity of the ketoglutarate dehydrogenase complex

The committed step in menaquinone biosynthesis is the formation of o-succinylbenzoate (OSB). It is presumed to require the reaction of a seven-carbon intermediate of the shikimate pathway with a succinic semialdehyde-thiamin pyrophosphate (TPP) anion, derived by decarboxylation of 2-ketoglutarate. The following evidence indicates that the decarboxylation is not a function of the ketoglutarate dehydrogenase complex but is carried out by a separate activity. (A) Cell-free extracts of Escherichia coli K12 without added TPP lose OSB synthase activity but retain all of the ketoglutarate dehydrogenase complex activities. (B) OSB synthase activity is inhibited by addition of tetrahydro-TPP (th-TPP) to the incubations. The ketoglutarate dehydrogenase complex activities are only inhibited by this analogue after an initial preincubation period. (C) The high molecular weight ketoglutarate dehydrogenase complex can be separated from OSB synthase activity by gel-permeation chromatography on Sepharose CL-6B. Experiment series A and B also provide supporting evidence that TPP does play an important role in menaquinone biosynthesis.     

Reaction kinetic pathway of the recombinant octaprenyl pyrophosphate synthase from Thermotoga maritima: how is it different from that of the mesophilic enzyme

Octaprenyl pyrophosphate synthase (OPPs) catalyzes the chain elongation of farnesyl pyrophosphate (FPP) via consecutive condensation reactions with five molecules of isopentenyl pyrophosphate (IPP) to generate all-trans C40-octaprenyl pyrophosphate. The polymer forms the side chain of ubiquinone that is involved in electron transport system to produce ATP. Our previous study has demonstrated that Escherichia coli OPPs catalyzes IPP condensation with a rate of 2 s(-1) but product release limits the steady-state rate at 0.02 s(-1) [Biochim. Biophys. Acta 1594 (2002) 64]. In the present studies, a putative gene encoding for OPPs from Thermotoga maritima, an anaerobic and thermophilic bacterium, was expressed, purified, and its kinetic pathway was determined. The enzyme activity at 25 degrees C was 0.005 s(-1) under steady-state condition and was exponentially increased with elevated temperature. In contrast to E. coli OPPs, IPP condensation rather than product release was rate limiting in enzyme reaction. The product of chain elongation catalyzed by T. maritima OPPs was C40 and the rate of its conversion to C45 was negligible. Under single-turnover condition with 10 microM OPPs-FPP complex and 1 microM IPP, only the C20 was formed rather than C20-C40 observed for E. coli enzyme. Together, our data suggest that the thermophilic OPPs from T. maritima has lower enzyme activity at 25 degrees C, higher product specificity, higher thermal stability and lower structural flexibility than its mesophilic counterpart from E. coli.     

Probing the mechanism of inactivation of human pyruvate dehydrogenase by phosphorylation of three sites

Activity of the mammalian pyruvate dehydrogenase complex (PDC) is regulated by phosphorylation-dephosphorylation of three serine residues (designated site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) in the alpha subunit of the pyruvate dehydrogenase (E1) component. Substitutions of the phosphorylation sites were generated by site-directed mutagenesis. Glutamate (S1E) and aspartate (S1D) substitutions at site 1 resulted in the complete loss of PDC activity; however, these mutants were variably active in the decarboxylation and 2,6-dichlorophenolindophenol assays. S1Q had only 3% of wild-type PDC activity. The apparent K(m) values for pyruvate increased for the mutants of site 1 when determined in the 2,6-dichlorophenolindophenol assay. The substitutions at sites 2 and 3 caused only moderate reductions in activity in the three assays. S3E had a 27-fold increase in the apparent K(m) for thiamine pyrophosphate and 8-fold increase in the K(i) for pyrophosphate. Site 3 was almost completely protected from phosphorylation by thiamine pyrophosphate. The results show that the size rather than negative charge of the substituted amino acid residue affects the active site of E1 and that modification of each of the three serine residues affect the active site in a site-specific manner for its ability to bind the cofactor and substrates.     

Partial purification and properties of CTP:phosphatidic acid cytidylyltransferase from membranes of Escherichia coli.

The cytosine liponucleotides CDP-diglyceride and dCDP-diglyceride are key intermediates in phospholipid biosynthesis in Escherichia coli (C. R. H. Raetz and E. P. Kennedy, J. Biol. Chem. 248:1098--1105, 1973). The enzyme responsible for their synthesis, CTP:phosphatidic acid cytidylytransferase, was solubilized from the cell envelope by a differential extraction procedure involving the detergent digitonin and was purified about 70-fold (relative to cell-free extracts) in the presence of detergent. In studies of the heat stability of the enzyme, activity decayed slowly at 63 degrees C. Initial velocity kinetic experiments suggested a sequential, rather than ping-pong, reaction mechanism; isotopic exchange reaction studies supported this conclusion and indicated that inorganic pyrophosphate is released before CDP-diglyceride in the reaction sequence. The enzyme utilized both CTP and dCTP as nucleotide substrate for the synthesis of CDP-diglyceride and dCDP-diglyceride, respectively. No distinction was observed between CTP and dCTP utilization in any of the purification, heat stability, and reaction mechanism studies. In addition, CTP and dCTP were competitive substrates for the partially purified enzyme. It therefore appears that a single enzyme catalyzes synthesis of both CDP-diglyceride and dCDP-diglyceride in E. coli. The enzyme also catalyzes a pyrophosphorolysis of CDP-diglyceride, i.e., the reverse of its physiologically important catalysis.     

Purification and characterization of indolepyruvate decarboxylase. A novel enzyme for indole-3-acetic acid biosynthesis in Enterobacter cloacae.

Indolepyruvate decarboxylase, a key enzyme for indole-3-acetic acid biosynthesis, was found in extracts of Enterobacter cloacae. The enzyme catalyzes the decarboxylation of indole-3-pyruvic acid to yield indole-3-acetaldehyde and carbon dioxide. The enzyme was purified to apparent homogeneity from Escherichia coli cells harboring the genetic locus for this enzyme obtained from E. cloacae. The results of gel filtration experiments showed that indolepyruvate decarboxylase is a tetramer with an M(r) of 240,000. In the absence of thiamine pyrophosphate and Mg2+, the active tetramers dissociate into inactive monomers and dimers. However, the addition of thiamine pyrophosphate and Mg2+ to the inactive monomers and dimers results in the formation of active tetramers. These results indicate that the thiamine pyrophosphate-Mg2+ complex functions in the formation of the tetramer, which is the enzymatically active holoenzyme. The enzyme exhibited decarboxylase activity with indole-3-pyruvic acid and pyruvic acid as substrates, but no decarboxylase activity was apparent with L-tryptophan, indole-3-lactic acid, beta-phenylpyruvic acid, oxalic acid, oxaloacetic acid, and acetoacetic acid. The Km values for indole-3-pyruvic acid and pyruvic acid were 15 microM and 2.5 mM, respectively. These results indicate that indole-3-acetic acid biosynthesis in E. cloacae is mediated by indolepyruvate decarboxylase, which has a high specificity and affinity for indole-3-pyruvic acid.     

alpha-Ketoglutarate dehydrogenase complex of Escherichia coli. A hybrid complex containing pyruvate dehydrogenase subunits from pyruvate dehydrogenase complex

The alpha-ketoglutarate dehydrogenase complex of Escherichia coli utilizes pyruvate as a poor substrate, with an activity of 0.082 units/mg of protein compared with 22 units/mg of protein for alpha-ketoglutarate. Pyruvate fully reduces the FAD in the complex and both alpha-keto[5-14C]glutarate and [2-14C]pyruvate fully [14C] acylate the lipoyl groups with approximately 10 nmol of 14C/mg of protein, corresponding to 24 lipoyl groups. NADH-dependent succinylation by [4-14C]succinyl-CoA also labels the enzyme with approximately 10 nmol of 14C/mg of protein. Therefore, pyruvate is a true substrate. However, the pyruvate and alpha-ketoglutarate activities exhibit different thiamin pyrophosphate dependencies. Moreover, 3-fluoropyruvate inhibits the pyruvate activity of the complex without affecting the alpha-ketoglutarate activity, and 2-oxo-3-fluoroglutarate inhibits the alpha-ketoglutarate activity without affecting the pyruvate activity. 3-Fluoro[1,2-14C]pyruvate labels about 10% of the E1 components (alpha-ketoacid dehydrogenases). The dihydrolipoyl transsuccinylase-dihydrolipoyl dehydrogenase subcomplex (E2E3) is activated as a pyruvate dehydrogenase complex by addition of E. coli pyruvate dehydrogenase, the E1 component of the pyruvate dehydrogenase complex. All evidence indicates that the alpha-ketoglutarate dehydrogenase complex purified from E. coli is a hybrid complex containing pyruvate dehydrogenase (approximately 10%) and alpha-ketoglutarate dehydrogenase (approximately 90%) as its E1 components.