A novel enzyme, citryl-CoA synthetase, catalysing the first step of the citrate cleavage reaction in Hydrogenobacter thermophilus TK-6

We attempted to purify ATP citrate lyase (ACL) from Hydrogenobacter thermophilus by following the citrate-, ATP- and CoA-dependent formation of an acyl-CoA species that was detected as hydroxamate. However, citryl-CoA rather than acetyl-CoA was found, indicating that the purified enzyme was a novel citryl-CoA synthetase (CCS) rather than ACL. Because the reaction catalysed by CCS corresponds to the first half of that mediated by ACL, CCS may be responsible for citrate cleavage in H. thermophilus. Thus, a novel citrate cleavage pathway, which does not involve ACL, appears to exist in this organism. Citryl-CoA synthetase is composed of two different polypeptides: a large beta subunit of 46 kDa and a small alpha subunit of 36 kDa. The corresponding genes were cloned and sequenced. The deduced amino acid sequences of the two subunits of CCS display significant similarity to those of succinyl-CoA synthetase (SCS) in the database. As a comparison, SCS was also purified from H. thermophilus and the corresponding genes were cloned and sequenced. Citryl-CoA synthetase and SCS were homologous, but showed different substrate specificity. The deduced amino acid sequences of the CCS subunits show similarity to part of the ACL sequence. The evolutionary relationship between CCS, SCS and ACL is discussed.     

Autotrophic CO2 fixation via the reductive tricarboxylic acid cycle in different lineages within the phylum Aquificae: evidence for two ways of citrate cleavage

Autotrophic carbon fixation was characterized in representative members of the three lineages of the bacterial phylum Aquificae. Enzyme activity measurements and the detection of key genes demonstrated that Aquificae use the reductive tricarboxylic acid (TCA) cycle for autotrophic CO(2) fixation. This is the first time that strains of the Hydrogenothermaceae and 'Desulfurobacteriaceae' have been investigated for enzymes of autotrophic carbon fixation. Unexpectedly, two different mechanisms of citrate cleavage could be identified within the Aquificae. Aquificaceae use citryl-CoA synthetase and citryl-CoA lyase, whereas Hydrogenothermaceae and 'Desulfurobacteriaceae' use ATP citrate lyase. The first mechanism is likely to represent the ancestral version of the reductive TCA cycle. Sequence analyses further suggest that ATP citrate lyase formed by a gene fusion of citryl-CoA synthetase and citryl-CoA lyase and subsequently became involved in a modified version of this pathway. However, rather than having evolved within the Aquificae, our phylogenetic analyses indicate that Aquificae obtained their ATP citrate lyase through lateral gene transfer. Aquificae play an important role in biogeochemical processes in a variety of high-temperature habitats. Thus, these findings substantiate the hypothesis that autotrophic carbon fixation through the reductive TCA cycle is widespread and contributes significantly to biomass production particularly in hydrothermal habitats.     

ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola is a heteromeric enzyme composed of two distinct gene products.

The reductive tricarboxylic acid cycle functions as a carbon dioxide fixation pathway in the green sulfur bacterium, Chlorobium limicola. ATP-citrate lyase, one of the key enzymes of this cycle, was partially purified from C. limicola strain M1 and the N-terminal sequence of a 65-kDa protein was found to show similarity toward eukaryotic ATP-citrate lyase. A DNA fragment was amplified with primers designed from this sequence and an internal sequence highly conserved among eukaryotic enzymes. Using this fragment as a probe, we isolated a DNA fragment containing two adjacent open reading frames, aclB (1197 bp) and aclA (1827 bp), whose products showed significant similarity to the N- and C-terminal regions of the human enzyme, respectively. Heterologous expression of these genes in Escherichia coli showed that both gene products were essential for ATP-citrate lyase activity. The recombinant enzyme was purified from the cell-free extract of E. coli harboring aclBA for further characterization. The molecular mass of the recombinant enzyme was determined to be approximately 532--557 kDa by gel-filtration. The enzyme catalyzed the cleavage of citrate in an ATP(-), CoA- and Mg(2+)-dependent manner, where ATP and Mg(2+) could be replaced by dATP and Mn(2+), respectively. ADP and oxaloacetate inhibited the reaction. These properties suggested that ATP-citrate lyase from C. limicola controlled the cycle flux depending on intracellular energy conditions. This paper provides the first direct evidence that a bacterial ATP-citrate lyase is a heteromeric enzyme, distinct from mammalian enzymes.     

Klebsiella pneumoniae genes for citrate lyase and citrate lyase ligase: localization, sequencing, and expression

In the course of studies on anaerobic citrate metabolism in Klebsiella pneumoniae, the DNA region upstream of the gene for the sodium-dependent citrate carrier (dtS) was investigated. Nucleotide sequence analysis revealed a cluster of five new genes that were oriented inversely to citS and probaby form an operon. The genes were named citCDEFG. Based on known protein sequence data, the gene products derived from citD, citE and citF could be identified as the λ-, β-, and α-subunits of citrate lyase, respectively. This enzyme catalyses the cleavage of citrate to oxaloacetate and acetate. The gene product derived from citC (calculated M_r 36476) exhibited no obvious similarity to other proteins. In the presence of acetate and ATP, cell extracts from a citC-expressing Escherichia coli strain were able to reactivate purified citrate lyase from K. pneumoniae that had been inactivated by chemical deacetylation of the prosthetic group. This represents 5-phosphoribosyi-dephospho-acetyl-coenzyme A which is covalently bound to serine-14 of the acyl carrier protein (λ-subunit). CitC was thus identified as acetate:SH-citrate lyase ligase. The function of the gene product derived from citG (M_r 32 645) has not yet been identified. Expression of the CitCDEFG gene cluster in E. coli led to the formation of citrate lyase which was active only in the presence of acetyl-coenzyme A, a compound known to substitute for the prosthetic group. These and other data strongly indicated that the enzyme synthesized in E. coli lacked its prosthetic group. Thus, additional genes besides citCDEFG appear to be required for the formation of holo-citrate lyase.     

Genetic enhancement of fatty acid synthesis by targeting rat liver ATP:citrate lyase into plastids of tobacco

ATP:citrate lyase (ACL) catalyzes the conversion of citrate to acetyl-coenzyme A (CoA) and oxaloacetate and is a key enzyme for lipid accumulation in mammals and oleaginous yeasts and fungi. To investigate whether heterologous ACL genes can be targeted and imported into the plastids of plants, a gene encoding a fusion protein of the rat liver ACL with the transit peptide for the small subunit of ribulose bisphosphate carboxylase was constructed and introduced into the genome of tobacco. This was sufficient to provide import of the heterologous protein into the plastids. In vitro assays of ACL in isolated plastids showed that the enzyme was active and synthesized acetyl-CoA. Overexpression of the rat ACL gene led to up to a 4-fold increase in the total ACL activity; this increased the amount of fatty acids by 16% but did not cause any major change in the fatty acid profile. Therefore, increasing the availability of acetyl-CoA as a substrate for acetyl-CoA carboxylase and subsequent reactions of fatty acid synthetase has a slightly beneficial effect on the overall rate of lipid synthesis in plants.     

ATP-citrate lyase from rat liver. Characterisation of the citryl-enzyme complexes.

The mechanism of ATP-citrate lyase has been proposed to involve a citryl-enzyme intermediate. When the enzyme is incubated with its substrates ATP and [14C]citrate, but in the absence of the final acceptor, two distinct types of citrate-containing complex can be isolated. At early time points, a highly unstable complex can be isolated by gel filtration which has a half-life of 36 s at 25 degrees C. This complex reacts rapidly with CoA, but cannot be acid-precipitated; behaviour consistent with its identification as enzyme-citryl phosphate. However, ATP-citrate lyase is also capable of undergoing a slow time-dependent covalent incorporation of radiolabel from [14C]citrate. This modification is acid-stable, non-specific, and cannot be reversed by the addition of CoA. When cytochrome is included in the reaction mixture as a heterologous acceptor, it is also citrylated. These reactions require that when ATP-citrate lyase is incubated with all its substrates except for CoA, a freely diffusible citrylating species is generated within the active site. This evidence suggests that there is no requirement for the mechanism of ATP-citrate lyase to proceed via a covalent citryl-enzyme intermediate. By analogy with succinyl-CoA synthetase, an enzyme which has a high degree of sequence similarity with ATP-citrate lyase, a simple mechanism is proposed for the enzyme in which citryl-CoA is produced by direct nucleophilic attack on citryl phosphate.     

The distinctiveness of ATP:citrate lyase from Aspergillus nidulans

ATP:citrate lyase (ACL), an important enzyme in lipid synthesis, has been purified from Aspergillus nidulans to a specific activity of 19.6 micromol min(-1) mg(-1), almost twice that of any other purified ACL and shown to be distinct from any previously purified ACL. The enzyme is a 371+/-31 kDa hexamer of 3 alpha, 3 beta proteins, unlike the 4 alpha tetramer found in rats or yeasts. The molecular weights of the alpha and beta protein subunits were determined by SDS-PAGE to be 70 and 55 kDa.ACL in A. nidulans (unlike Aspergillus niger) appears to be regulated by the carbon source present in the media. In crude extracts, it was found at high activity (88 micromol min(-1) mg protein(-1)) in glucose-grown cells but only at low activity (10 micromol min(-1) mg protein(-1)) in acetate-grown cells.     

Purification and properties of a glucuronan lyase from Sinorhizobium meliloti M5N1CS (NCIMB 40472).

A glucuronan lyase extracted from Sinorhizobium meliloti strain M5N1CS was purified to homogeneity by anion-exchange chromatography. The purified enzyme corresponds to a monomer with a molecular mass of 20 kDa and a pI of 4.9. A specific activity was found only for polyglucuronates leading to the production of 4,5-unsaturated oligoglucuronates. The enzyme activity was optimal at pH 6.5 and 50 degrees C. Zn(2+), Cu(2+), and Hg(2+) (1 mM) inhibited the enzyme activity. No homology of the enzyme N-terminal amino acid sequence was found with any of the previously published protein sequences. This enzyme purified from S. meliloti strain M5N1CS corresponding to a new lyase was classified as an endopolyglucuronate lyase.     

Studies on yeast peroxisomal citrate synthase

Peroxisomal (nonmitochondrial) citrate synthase (CS2) has been purified from a Saccharomyces cerevisiae strain in which the gene for the mitochondrial citrate synthase (CS1) had been disrupted and no CS1 protein is produced. The enzyme, CS2, the sequence of which had been previously determined from its DNA, behaved differently from CS1 in its purification, kinetics, stability, and binding to the inner surface of mitochondrial inner membranes.     

Conversion, by limited proteolysis, of an archaebacterial citrate synthase into essentially a citryl-CoA hydrolase.

1. Limited proteolysis of citrate synthase from Sulfolobus solfataricus by trypsin reduced the rate of the overall reaction (acetyl-CoA + oxaloacetate + H2O----citrate + CoASH) to 4% but did not affect the hydrolysis of citryl-CoA. Experimental results indicate that a connecting link between the enzyme's ligase and hydrolase activity becomes impaired specifically on treatment with trypsin. Other proteolytic enzymes like chymotrypsin and subtilisin inactivated catalytic functions of citrate synthase, ligase and hydrolase, equally well. 2. Tryptic hydrolysis occurs at the N-terminal region of citrate synthase, but a study by SDS/PAGE revealed no difference in molecular mass between native and proteolytically nicked citrate synthase. The peptide removed from the enzyme by trypsin, therefore, contains less than about 15 amino acid residues. 3. The Km values of the substrates for both native and nicked enzyme were identical, as was the state of aggregation (dimeric) of the two enzyme species. These could be separated by affinity chromatography on Blue-Sepharose and differentiated by their isoelectric points (pI = 6.68 +/- 0.08 and pI = 6.37 +/- 0.03 for native citrate synthase and the large tryptic peptide, respectively) as well as by the N-terminus which is blocked in the native enzyme only. 4. Edman degradation of the large tryptic fragment yielded the N-terminal sequence GLEDVYIKSTSLTYIDGVNGVLRY, which is 71% identical to the N-terminal region (positions 9-32) of citrate synthase from Thermoplasma acidophilum. 5. The conversion of citrate synthase into essentially a citryl-CoA hydrolase is considered the consequence of a conformational change thought to occur on tryptic removal of the N-terminal small peptide.     

ATP citrate lyase in the glaucocystophyte alga Cyanophora paradoxa is a cytosolic enzyme: characterisation of the gene for the large subunit at the cDNA and genomic levels

The single-copy gene for the large subunit of ATP citrate lyase (ACL) from the alga Cyanophora paradoxa was characterized at the cDNA and genome levels. The gene product showed high sequence similarity to its mammalian counterparts, but is smaller in size, as is also found for the fungal subunit Acl1 and, most probably, for the corresponding subunit from Arabidopsis thaliana. The C. paradoxa gene is interrupted by at least 12 introns of 53-65 bp with conserved border and branchpoint sequences, and the product lacks a stroma-targeting peptide. Enzyme activity was found in the cytosol of C. paradoxa but not in the plastid (cyanelle) fraction. This is in contrast to the subcellular distribution of ATP citrate lyases in higher plants, where both chloroplast and cytosolic enzymes have been reported.     

Purification and some properties of the citrate synthase from a marine Pseudomonas.

Citrate synthase (citrate-oxaloacetate lyase (CoA acetylating), EC 4.1.3.7) has been purified to electrophoretic homogeneity from a marine Pseudomonas. The enzyme was made up of identical subunits, with a molecular wieght of about 53 000, as determined by sodium dodecyl sulphate - polyacrylamide gel electrophoresis. The native enzyme (citrate synthase II, CS II) could be dissociated by dialysis against 20 mM phosphate (Pi), pH 7; the enzyme thus obtained (citrate synthase I, CS I) was still active, but presented different molecular weight and kinetic and regulatory properties. CS II was activated by adenosine monophosphate (AMP), Pi, and KCl, and inhibited by reduced nicotinamide adenine dinucleotide (NADH), being apparently insensitive to adenosine triphosphate (ATP) and adenosine diphosphate (ADP). The inhibition by NADH was completely counteracted by 0.1 mM AMP, but not by 50 mM Pi or 0.1 M KCl. The activation by KCl and Pi, or by KCl and AMP was nearly additive, whereas that by AMP and Pi was not. The activators acted essentially by increasing Vmax, although they also caused a decrease in the Km values. CS I was inhibited by ATP, ADP, AMP, and KCl, and was insensitive to NADH. CS I could be reassociated after elimination of Pi by dialysis, regaining the higher molecular weight and the activation by AMP characteristic of CS II.     

Photoaffinity labeling of Klebsiella aerogenes citrate lyase by p-azidobenzoyl coenzyme A

p-Azidobenzoyl coenzyme A functions as a linear competitive inhibitor for (3S)-citryl-CoA in the citryl-CoA oxaloacetate-lyase reaction catalyzed by the Klebsiella aerogenes deacetylcitrate lyase complex (Ki = 80 microM; (3S)-citryl-CoA Km = 67 microM). Inactivation is irreversible on photolysis of p-azidobenzoyl-CoA in the presence of the deacetylcitrate lyase complex. Mg2+ is not required for the inactivation. Inactivation is blocked by (3S)-citryl-CoA in the presence of ethylenediaminetetraacetic acid. p-Azidobenzoyl-CoA has no effect on the acetyl-CoA:citrate CoA transferase activity of both the deacetylcitrate lyase complex and its isolated transferase subunit. The stoichiometry of the CoA ester binding has been investigated by the use of p-azido[14C]benzoyl-CoA as a photoaffinity reagent. The labeling is exclusively on the lyase beta subunit of the citrate lyase complex.     

Characterization of ATP citrate lyase from Chlorobium limicola.

ATP citrate lyase (EC 4.1.3.8) from Chlorobium limicola was partially purified. It was established that the consumption of substrates and the formation of products proceeded stoichiometrically and that citrate cleavage was of the si-type. ADP and oxaloacetate inhibited enzyme activity. Oxaloacetate also inhibited the growth of C. limicola.     

Identification of a gene cluster in Klebsiella pneumoniae which includes citX, a gene required for biosynthesis of the citrate lyase prosthetic group

The biosynthesis of the 2'-(5"-phosphoribosyl)-3'-dephospho-coenzyme A (CoA) prosthetic group of citrate lyase (EC 4.1.3.6), a key enzyme of citrate fermentation, proceeds via the initial formation of the precursor 2'-(5"-triphosphoribosyl)-3'-dephospho-CoA and subsequent transfer to apo-citrate lyase with removal of pyrophosphate. In Escherichia coli, the two steps are catalyzed by CitG and CitX, respectively, and the corresponding genes are part of the citrate lyase gene cluster, citCDEFXG. In the homologous citCDEFG operon of Klebsiella pneumoniae, citX is missing. A search for K. pneumoniae citX led to the identification of a second genome region involved in citrate fermentation which comprised the citWX genes and the divergent citYZ genes. The citX gene was confirmed to encode holo-citrate lyase synthase, whereas citW was shown to encode a citrate carrier, the third one identified in this species. The citYZ genes were found to encode a two-component system consisting of the sensor kinase CitY and the response regulator CitZ. Remarkably, both proteins showed >or=40% sequence identity to the citrate-sensing CitA-CitB two-component system, which is essential for the induction of the citrate fermentation genes in K. pneumoniae. A citZ insertion mutant was able to grow anaerobically with citrate, indicating that CitZ is not essential for expression of citrate fermentation genes. CitX synthesis was induced to a basal level under anaerobic conditions, independent of citrate, CitB, and CitZ, and to maximal levels during anaerobic growth with citrate as the sole carbon source. Similar to the other citrate fermentation enzymes, CitX synthesis was apparently subject to catabolite repression.     

Polysaccharide lyase: molecular cloning, sequencing, and overexpression of the xanthan lyase gene of Bacillus sp. strain GL1

When grown on xanthan as a carbon source, the bacterium Bacillus sp. strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H. Nankai, W. Hashimoto, H. Miki, S. Kawai, and K. Murata, Appl. Environ. Microbiol. 65:2520-2526, 1999). A gene for the lyase was cloned, and its nucleotide sequence was determined. The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide with a molecular weight of 99,308. The polypeptide had a signal peptide (2 kDa) consisting of 25 amino acid residues preceding the N-terminal amino acid sequence of the enzyme and exhibited significant homology with hyaluronidase of Streptomyces griseus (identity score, 37.7%). Escherichia coli transformed with the gene without the signal peptide sequence showed a xanthan lyase activity and produced intracellularly a large amount of the enzyme (400 mg/liter of culture) with a molecular mass of 97 kDa. During storage at 4 degrees C, the purified enzyme (97 kDa) from E. coli was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to those of the enzyme (75 kDa) from Bacillus sp. strain GL1, specifically in optimum pH and temperature for activity, substrate specificity, and mode of action. Logarithmically growing cells of Bacillus sp. strain GL1 on the medium with xanthan were also found to secrete not only xanthan lyase (75 kDa) but also a 97-kDa protein with the same N-terminal amino acid sequence as that of xanthan lyase (75 kDa). These results suggest that, in Bacillus sp. strain GL1, xanthan lyase is first synthesized as a preproform (99 kDa), secreted as a precursor (97 kDa) by a signal peptide-dependent mechanism, and then processed into a mature form (75 kDa) through excision of a C-terminal protein fragment with a molecular mass of 22 kDa.     

Both subunits of ATP-citrate lyase from Chlorobium tepidum contribute to catalytic activity

ATP-citrate lyase (ACL) is an essential enzyme of the reductive tricarboxylic acid (RTCA) pathway of CO(2) assimilation. The RTCA pathway occurs in several groups of autotrophic prokaryotes, including the green sulfur bacteria. ACL catalyzes the coenzyme A (CoA)-dependent and MgATP-dependent cleavage of citrate into oxaloacetate and acetyl-CoA, representing a key step in the RTCA pathway. To characterize this enzyme from the green sulfur bacterium Chlorobium tepidum and determine the role of its two distinct polypeptide chains, recombinant holo-ACL as well as its two individual subunit polypeptides were synthesized in Escherichia coli. The recombinant holoenzyme, prepared from coexpressed large and small ACL genes, and the individual large and small subunit polypeptides, prepared from singly expressed genes, were all purified to homogeneity to high yield. Purified recombinant holo-ACL was isolated at high specific activity, and its k(cat) was comparable to that of previously prepared native C. tepidum ACL. Moreover, the purified recombinant large and small subunit polypeptides were able to reconstitute the holo-ACL in vitro, with activity levels approaching that of recombinant holo-ACL prepared from coexpressed genes. Stoichiometric amounts of each subunit protein were required to maximize the activity and form the most stable structure of reconstituted holo-ACL. These results suggested that this reconstitution system could be used to discern the catalytic role of specific amino acid residues on each subunit. Reconstitution and mutagenesis studies together indicated that residues of each subunit contributed to different aspects of the catalytic mechanism, suggesting that both subunit proteins contribute to the active site of C. tepidum ACL.     

A role for ATP-citrate lyase, malic enzyme, and pyruvate/citrate cycling in glucose-induced insulin secretion

In pancreatic beta-cells, metabolic coupling factors generated during glucose metabolism and pyruvate cycling through anaplerosis/cataplerosis processes contribute to the regulation of insulin secretion. Pyruvate/citrate cycling across the mitochondrial membrane leads to the production of malonyl-CoA and NADPH, two candidate coupling factors. To examine the implication of pyruvate/citrate cycling in glucose-induced insulin secretion (GIIS), different steps of the cycle were inhibited in INS 832/13 cells by pharmacological inhibitors and/or RNA interference (RNAi) technology: mitochondrial citrate export, ATP-citrate lyase (ACL), and cytosolic malic enzyme (ME1). The inhibitors of the di- and tri-carboxylate carriers, n-butylmalonate and 1,2,3-benzenetricarboxylate, respectively, reduced GIIS, indicating the importance of transmitochondrial transport of tri- and dicarboxylates in the action of glucose. To directly test the role of ACL and ME1 in GIIS, small hairpin RNA (shRNA) were used to selectively decrease ACL or ME1 expression in transfected INS 832/13 cells. shRNA-ACL reduced ACL protein levels by 67%, and this was accompanied by a reduction in GIIS. The amplification/K(ATP)-independent pathway of GIIS was affected by RNAi knockdown of ACL. The ACL inhibitor radicicol also curtailed GIIS. shRNA-ME1 reduced ME1 activity by 62% and decreased GIIS. RNAi suppression of either ACL or ME1 did not affect glucose oxidation. However, because ACL is required for malonyl-CoA formation, inhibition of ACL expression by shRNA-ACL decreased glucose incorporation into palmitate and increased fatty acid oxidation in INS 832/13 cells. Taken together, the results underscore the importance of pyruvate/citrate cycling in pancreatic beta-cell metabolic signaling and the regulation of GIIS.     

Citrate lyase from Streptococcus diacetilactis

Citrate lyase (EC 4.1.3.6) was purified 38-fold from cell-free extracts of Streptococcus diacetilactis. The enzyme was homogeneous in analytical ultracentrifugation and polyacrylamide gel electrophoresis The final enzyme preparation contained acetate: HS-citrate lyase ligase—an acetylating enzyme which converts inactive HS-citrate lyase into enzymatically active acetyl-S-citrate lyase. This enzyme activity was purified 25-fold over the crude extract and seemed to be associated with citrate lyase. Partially purified citrate lyase from Leuconostoc citrovorum contained also its acetylating enzyme. Purified citrate lyases from Klebsiella aerogenes and Rhodopseudomonas gelatinosa were devoid of acetylating enzyme activity. The HS-form of citrate lyase from S. diacetilactis was completely acetylated and hence activated by incubation with ATP and acetate for 25 min at 25° C. The enzyme did not acetylate the HS-lyases from R. gelatinosa and K. aerogenes. In contrast to the citrate lyases from R. gelatinosa and K. aerogenes the enzymes from S. diacetilactis and L. citrovorum showed onlya very weak reaction inactivation. It is assumed that this is due to the association of the acetylating enzymes with these lyases.     

Hysteretic behaviour of citrate synthase. The reaction mechanism and the exclusion of synthase being a hysteretic enzyme

The non-Michaelis-Menten kinetics, burst and steady-state periods, expressed by citrate synthase in the presence of citryl-CoA, were investigated by labelling experiments with trace amounts of [14C]acetyl-CoA. The results indicate that citrate becomes labelled in the reaction of liberated acetyl-CoA with the binary synthase.oxaloacetate complex that is transiently generated in the lyase reaction of citryl-CoA. Mediated by the hydrolase function of synthase, the counteracting citryl-CoA lyase and ligase reactions operate towards a transient flow equilibrium. This precedes the thermodynamic equilibrium and is established during the burst period; it is maintained under steady-state conditions and corresponds to the formation of transiently nonproductive synthase. The rates of both synthase partial reactions, therefore, are likewise affected. Oxaloacetate in the presence of acetyl-CoA competitively inhibits the hydrolysis of citryl-CoA and vice versa. In the synthase dependence of the burst periods and during the time dependence of the steady-state periods, nonproportionally more of physiological substrates participate in citrate formation. The nonproportional increase is a consequence of the continuously changing conditions to establish or to maintain the flow equilibrium, respectively, during the reaction progress. Third rate periods after the steady state result if the equilibrium conditions cannot be satisfied. High concentrations of oxaloacetate inhibit the expression of non-Michaelis-Menten kinetics by formation of nonproductive synthase.oxaloacetate complex. The supply of acetyl-CoA is then sufficient and the formation of the flow equilibrium prevented. The implication of the results with structural work is discussed.