A new spectrophotometric assay for citrate synthase and its use to assess the inhibitory effects of palmitoyl thioesters.
ABSTRACT: We have demonstrated that citrate synthase may be assayed by a simple, discontinuous, spectrophotometric procedure based on the measurement of oxaloacetate utilization with 2,4-dinitrophenylhydrazine. The assay is applicable both to the purified enzyme and to cell extracts, and has the advantage that it can be used in the presence of high concentrations of thiols and thioesters. We have used this new assay in part of our investigations into the inhibitory effects of palmitoyl thioesters on diverse citrate synthases. Both palmitoyl-CoA and palmitoyl thioglycollate inhibit citrate synthases from pig heart, Bacillus megaterium and Escherichia coli, the E. coli enzyme showing the greatest sensitivity to these effectors. With palmitoyl-CoA the extent of inhibition is time-dependent, but the enzymes can be protected from the effect by the substrates oxaloacetate and acetyl-CoA. Using the dinitrophenylhydrazine assay, we have shown that the thioester bond is essential for inhibition; that is, if the palmitoyl thioesters are cleaved to give a mixture of palmitate and a thiol compound, the inhibitions of pig heart and B. megaterium citrate synthases are eliminated and that of the E. coli enzyme is markedly decreased.
Project description:A simple statistical approach was used to generate predictive models of the proteolysis of multisubunit enzymes in order to correlate the loss of enzyme activity with the loss of native subunit. The models were applied to the trypsinolysis of the citrate synthases of pig heart, Bacillus megaterium and Escherichia coli. With the dimeric citrate synthases (pig heart and B. megaterium) trypsinolysis of one of the subunits appears to destroy the activity of the whole enzymic molecule. The hexameric E. coli citrate synthase behaves like a trimer of dimeric units, each of the dimers behaving similarly to the B. megaterium and pig heart enzymes. Palmitoyl-CoA is required for the trypsinolysis of pig heart citrate synthase, and at relatively high concentrations of this compound trypsinolysis of one subunit leaves the other subunit fully active. Palmitoyl-CoA is not required for the trypsinolysis of the other citrate synthases, and high concentrations of this metabolite do not affect the correlation of proteolysis with inactivation of these enzymes.
Project description:The first step in the overall catalytic mechanism of citrate synthase is the binding and polarization of oxaloacetate. Active-site residues Arg-314, Asp-312 and His-264 in Escherichia coli citrate synthase, which are involved in oxaloacetate binding, were converted by site-directed mutagenesis to Gln-314, Asn-312 and Asn-264 respectively. The R314Q and D312N mutants expressed negligible overall catalytic activity at pH 8.0, the normal assay pH, but substantial activities for the partial reactions that reflect the cleavage and hydrolysis of the substrate intermediate citryl-CoA. However, when the pH was lowered to 7.0, the overall reaction of the mutants became significant, in contrast to the wild-type enzyme, whereas the two mutants exhibited reduced activities for the partial reactions. This result is consistent with the existence of a rate-limiting step between the two partial reactions for these mutants that is pH-dependent. The Km for oxaloacetate for the two mutants was increased 10-fold and was paralleled by an increase in the Km for citryl-CoA, whereas the Km for acetyl-CoA was increased only 2-fold. Overall, there was a striking parallel between the results obtained for these two mutants, which suggests that they are functionally linked in the E. coli enzyme. The equivalent of these two residues form a salt bridge in the pig heart citrate synthase crystal structure. The H264N mutant, in which the amide nitrogen of asparagine should mimic the delta-nitrogen of histidine, showed negligible activity in terms of both overall and partial catalysis, which may result from a hindrance of conformational change upon oxaloacetate binding. The affinity of this mutant for oxaloacetate appeared to be greatly reduced when investigated using indirect fluorescence and chemical modification techniques.
Project description:Citrate synthase was purified to homogeneity from a Gram-positive bacterium (Bacillus megaterium) for the first time. The Mr of the native enzyme was determined to be 84 000 (S.E.M. +/- 5000). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration in guanidinium chloride revealed a single protein species of Mr 40 300 (S.E.M. +/- 4400), indicating a dimeric enzyme. This dimeric structure was confirmed by cross-linking the native enzyme with dimethyl suberimidate and with glutaraldehyde, followed by electrophoretic analysis. The enzyme follows Michaelis-Menten kinetics with respect to both substrates, acetyl-CoA and oxaloacetate, and is sensitive to non-specific inhibition by a range of adenine nucleotides. In both molecular and catalytic properties the citrate synthase closely resembles the enzyme from eukaryotic sources and contrasts markedly with the larger, hexameric, enzyme from Gram-negative bacteria.
Project description:Glutamate is usually synthesized from acetyl coenzyme A (acetyl-CoA) via citrate, isocitrate, and 2-oxoglutarate. Genome analysis revealed that in Syntrophus aciditrophicus, the gene for Si-citrate synthase is lacking. An alternative pathway starting from the catabolic intermediate glutaconyl-CoA via 2-hydroxyglutarate could be excluded by genomic analysis. On the other hand, a putative gene (SYN_02536; NCBI gene accession no. CP000252.1) annotated as coding for isopropylmalate/citramalate/homocitrate synthase has been shown to share 49% deduced amino acid sequence identity with the gene encoding Re-citrate synthase of Clostridium kluyveri. We cloned and overexpressed this gene in Escherichia coli together with the genes encoding the chaperone GroEL. The recombinant homotetrameric enzyme with a C-terminal Strep-tag (4 × 72,892 Da) was separated from GroEL on a Strep-Tactin column by incubation with ATP, K(+), and Mg(2+). The pure Re-citrate synthase used only acetyl-CoA and oxaloacetate as the substrates. As isolated, the enzyme contained stoichiometric amounts of Ca(2+) (0.9 Ca/73 kDa) but achieved higher specific activities in the presence of Mn(2+) (1.2 U/mg) or Co(2+) (2.0 U/mg). To determine the stereospecificity of the enzyme, [(14)C]citrate was enzymatically synthesized from oxaloacetate and [1-(14)C]acetyl-CoA; the subsequent cleavage by Si-citrate lyase yielded unlabeled acetate and labeled oxaloacetate, demonstrating that the enzyme is a Re-citrate synthase. The production of Re-citrate synthase by S. aciditrophicus grown axenically on crotonate was revealed by synthesis of [(14)C]citrate in a cell extract followed by stereochemical analysis. This result was supported by detection of transcripts of the Re-citrate synthase gene in axenic as well as in syntrophic cultures using quantitative reverse transcriptase PCR (qRT-PCR).
Project description:Substrate-depleted rat liver mitochondria will reaccumulate malate, succinate, oxoglutarate, beta-hydroxybutyrate and glutamate if provided with an energy source and Ca(2+) (or Ca(2+) and Mn(2+)). The energy requirement for ion uptake by fresh mitochondria causes a transient oxidation of their NADH and presumably this leads to an increased oxaloacetate concentration. A consequence is the promotion of formation of citrate, which tends to remain in the particles, provided the pH is above 7. Analyses made of systems blocked with fluorocitrate show that citrate accumulates when Ca(2+) is added with the following substrates; (a) pyruvate in the presence of ATP or malate, (b) palmitoyl-l(-)-carnitine in presence of malate and (c) oxoglutarate. Lowering the pH, even to 6.8, causes the citrate to emerge. This could be the basis of a cellular control mechanism. The generation of citrate in response to Ca(2+) can explain the stoichiometry of one proton ejected per Ca(2+) ion taken up. The new carboxyl group formed from acetyl-CoA when it condenses with oxaloacetate provides an internal anionic charge and a proton to emerge when Ca(2+) enters.
Project description:Citrate synthase (CS) performs two half-reactions: the mechanistically intriguing condensation of acetyl-CoA with oxaloacetate (OAA) to form citryl-CoA and the subsequent, slower hydrolysis of citryl-CoA that generally dominates steady-state kinetics. The condensation reaction requires the abstraction of a proton from the methyl carbon of acetyl-CoA to generate a reactive enolate intermediate. The carbanion of that intermediate then attacks the OAA carbonyl to furnish citryl-CoA, the initial product. Using stopped-flow and steady-state fluorescence methods, kinetic substrate isotope effects, and mutagenesis of active site residues, we show that all of the processes that occur in the condensation half-reaction performed by Thermoplasma acidophilum citrate synthase (TpCS) with the natural thioester substrate, acetyl-CoA, also occur with the ketone inhibitor dethiaacetyl-CoA. Free energy profiles demonstrate that the nonhydrolyzable product of the condensation reaction, dethiacitryl-CoA, forms a particularly stable complex with TpCS but not pig heart CS.
Project description:The genome annotations of all sequenced Dehalococcoides strains lack a citrate synthase, although physiological experiments have indicated that such an activity should be encoded. We here report that a Re face-specific citrate synthase is synthesized by Dehalococcoides strain CBDB1 and that this function is encoded by the gene cbdbA1708 (NCBI accession number CAI83711), previously annotated as encoding homocitrate synthase. Gene cbdbA1708 was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified. The enzyme catalyzed the condensation of oxaloacetate and acetyl coenzyme A (acetyl-CoA) to citrate. The protein did not have homocitrate synthase activity and was inhibited by citrate, and Mn2+ was needed for full activity. The stereospecificity of the heterologously expressed citrate synthase was determined by electrospray ionization liquid chromatography-mass spectrometry (ESI LC/MS). Citrate was synthesized from [2-(13)C]acetyl-CoA and oxaloacetate by the Dehalococcoides recombinant citrate synthase and then converted to acetate and malate by commercial citrate lyase plus malate dehydrogenase. The formation of unlabeled acetate and 13C-labeled malate proved the Re face-specific activity of the enzyme. Shotgun proteome analyses of cell extracts of strain CBDB1 demonstrated that cbdbA1708 is expressed in strain CBDB1.
Project description:Members of the family Geobacteraceae are commonly the predominant Fe(III)-reducing microorganisms in sedimentary environments, as well as on the surface of energy-harvesting electrodes, and are able to effectively couple the oxidation of acetate to the reduction of external electron acceptors. Citrate synthase activity of these organisms is of interest due to its key role in acetate metabolism. Prior sequencing of the genome of Geobacter sulfurreducens revealed a putative citrate synthase sequence related to the citrate synthases of eukaryotes. All citrate synthase activity in G. sulfurreducens could be resolved to a single 49-kDa protein via affinity chromatography. The enzyme was successfully expressed at high levels in Escherichia coli with similar properties as the native enzyme, and kinetic parameters were comparable to related citrate synthases (kcat= 8.3 s(-1); Km= 14.1 and 4.3 microM for acetyl coenzyme A and oxaloacetate, respectively). The enzyme was dimeric and was slightly inhibited by ATP (Ki= 1.9 mM for acetyl coenzyme A), which is a known inhibitor for many eukaryotic, dimeric citrate synthases. NADH, an allosteric inhibitor of prokaryotic hexameric citrate synthases, did not affect enzyme activity. Unlike most prokaryotic dimeric citrate synthases, the enzyme did not have any methylcitrate synthase activity. A unique feature of the enzyme, in contrast to citrate synthases from both eukaryotes and prokaryotes, was a lack of stimulation by K+ ions. Similar citrate synthase sequences were detected in a diversity of other Geobacteraceae members. This first characterization of a eukaryotic-like citrate synthase from a prokaryote provides new insight into acetate metabolism in Geobacteraceae members and suggests a molecular target for tracking the presence and activity of these organisms in the environment.
Project description:1. Acetyl-CoA carboxylase activity was measured in extracts of rat epididymal fat-pads either on preparation of the extracts (initial activity) or after incubation of the extracts with citrate (total activity). In the presence of glucose or fructose, brief exposure of pads to insulin increased the initial activity of acetyl-CoA carboxylase; no increase occurred in the absence of substrate. Adrenaline in the presence of glucose and insulin decreased the initial activity. None of these treatments led to a substantial change in the total activity of acetyl-CoA carboxylase. A large decrease in the initial activity of acetyl-CoA carboxylase also occurred with fat-pads obtained from rats that had been starved for 36h although the total activity was little changed by this treatment. 2. Conditions of high-speed centrifugation were found which appear to permit the separation of the polymeric and protomeric forms of the enzyme in fat-pad extracts. After the exposure of the fat-pads to insulin (in the presence of glucose), the proportion of the enzyme in the polymeric form was increased, whereas exposure to adrenaline (in the presence of glucose and insulin) led to a decrease in enzyme activity. 3. These changes are consistent with a role of citrate (as activator) or fatty acyl-CoA thioesters (as inhibitors) in the regulation of the enzyme by insulin and adrenaline; no evidence that the effects of these hormones involve phosphorylation or dephosphorylation of the enzyme could be found. 4. Changes in the whole tissue concentration of citrate and fatty acyl-CoA thioesters were compared with changes in the initial activity of acetyl-CoA carboxylase under a variety of conditions of incubation. No correlation between the citrate concentration and the initial enzyme activity was evident under any condition studied. Except in fat-pads which were exposed to insulin there was little inverse correlation between the concentration in the tissue of fatty acyl-CoA thioesters and the initial activity of acetyl-CoA carboxylase. 5. It is suggested that changes in the concentration of free fatty acyl-CoA thioesters (which may not be reflected in whole tissue concentrations of these metabolites) may be important in the regulation of the activity of acetyl-CoA carboxylase. The possibility is discussed that the concentration of free fatty acyl-CoA thioesters may be controlled by binding to a specific protein with properties similar to albumin.
Project description:Studies of citrate synthase (CitA) were carried out to investigate its role in morphological development and biosynthesis of antibiotics in Streptomyces coelicolor. Purification of CitA, the major vegetative enzyme activity, allowed characterization of its kinetic properties. The apparent K(m) values of CitA for acetyl coenzyme A (acetyl-CoA) (32 microM) and oxaloacetate (17 microM) were similar to those of citrate synthases from other gram-positive bacteria and eukaryotes. CitA was not strongly inhibited by various allosteric feedback inhibitors (NAD(+), NADH, ATP, ADP, isocitrate, or alpha-ketoglutarate). The corresponding gene (citA) was cloned and sequenced, allowing construction of a citA mutant (BZ2). BZ2 was a glutamate auxotroph, indicating that citA encoded the major citrate synthase allowing flow of acetyl-CoA into the tricarboxylic acid (TCA) cycle. Interruption of aerobic TCA cycle-based metabolism resulted in acidification of the medium and defects in morphological differentiation and antibiotic biosynthesis. These developmental defects of the citA mutant were in part due to a glucose-dependent medium acidification that was also exhibited by some other bald mutants. Unlike other acidogenic bald strains, citA and bldJ mutants were able to produce aerial mycelia and pigments when the medium was buffered sufficiently to maintain neutrality. Extracellular complementation studies suggested that citA defines a new stage of the Streptomyces developmental cascade.