InChI=1S/C23H38N7O18P3S/c1- 23(2,18(35)21(36)26-4-3-13(31)25-5-6-52-8-14(32)33)9-45-51(42,43)48-50(40,41)44-7-12-17(47-49(37,38)39)16(34)22(46-12)30-11-29-15-19(24)27-10-28-20(15)30/h10-12,16-18,22,34-35H,3-9H2,1-2H3,(H,25,31)(H,26,36)(H,32,33)(H,40,41)(H,42,43)(H2,24,27,28)(H2,37,38,39)/t12-,16-,17-,18+,22-/m1/s1 |
| OBUOSIHPWVNVJN-GRFIIANRSA-N |
| [C@@H]1(N2C3=C(C(=NC=N3)N)N=C2)O[C@H](COP(OP(OCC([C@H](C(NCCC(NCCSCC(O)=O)=O)=O)O)(C)C)(=O)O)(=O)O)[C@H]([C@H]1O)OP(O)(O)=O |
|
|
Brassica napus metabolite
Any plant metabolite that is produced by rapeseed (Brassica napus).
EC 2.3.3.1 [citrate (Si)-synthase] inhibitor
An EC 2.3.3.* (acyltransferase converting acyl to alkyl group on transfer) inhibitor that interferes with the action of citrate (Si)-synthase, EC 2.3.3.1.
|
|
View more via ChEBI Ontology
|
Carboxymethyl-coenzyme A
|
ChemIDplus
|
|
S-(carboxymethyl)-CoA
|
ChEBI
|
|
S-(carboxymethyl)-coenzyme A
|
ChEBI
|
|
79955-48-9
|
CAS Registry Number
|
ChemIDplus
|
Duckworth HW, Nguyen NT, Gao Y, Donald LJ, Maurus R, Ayed A, Bruneau B, Brayer GD (2013)
Enzyme-substrate complexes of allosteric citrate synthase: evidence for a novel intermediate in substrate binding.
Biochimica et biophysica acta 1834, 2546-2553 [PubMed:23954305]
[show Abstract]
The citrate synthase (CS) of Escherichia coli is an allosteric hexameric enzyme specifically inhibited by NADH. The crystal structure of wild type (WT) E. coli CS, determined by us previously, has no substrates bound, and part of the active site is in a highly mobile region that is shifted from the position needed for catalysis. The CS of Acetobacter aceti has a similar structure, but has been successfully crystallized with bound substrates: both oxaloacetic acid (OAA) and an analog of acetyl coenzyme A (AcCoA). We engineered a variant of E. coli CS wherein five amino acids in the mobile region have been replaced by those in the A. aceti sequence. The purified enzyme shows unusual kinetics with a low affinity for both substrates. Although the crystal structure without ligands is very similar to that of the WT enzyme (except in the mutated region), complexes are formed with both substrates and the allosteric inhibitor NADH. The complex with OAA in the active site identifies a novel OAA-binding residue, Arg306, which has no functional counterpart in other known CS-OAA complexes. This structure may represent an intermediate in a multi-step substrate binding process where Arg306 changes roles from OAA binding to AcCoA binding. The second complex has the substrate analog, S-carboxymethyl-coenzyme A, in the allosteric NADH-binding site and the AcCoA site is not formed. Additional CS variants unable to bind adenylates at the allosteric site show that this second complex is not a factor in positive allosteric activation of AcCoA binding. | Kurz LC, Shah S, Frieden C, Nakra T, Stein RE, Drysdale GR, Evans CT, Srere PA (1995)
Catalytic strategy of citrate synthase: subunit interactions revealed as a consequence of a single amino acid change in the oxaloacetate binding site.
Biochemistry 34, 13278-13288 [PubMed:7577912]
[show Abstract]
The active site of pig heart citrate synthase contains a histidine residue (H320) which interacts with the carbonyl oxygen of oxaloacetate and is implicated in substrate activation through carbonyl bond polarization, a major catalytic strategy of the enzyme. We report here the effects on the catalytic mechanism of changing this important residue to glycine. H320G shows modest impairment in substrate Michaelis constants [(7-16)-fold] and a large decrease in catalysis (600-fold). For the native enzyme, the chemical intermediate, citryl-CoA, is both hydrolyzed and converted back to reactants, oxaloacetate and acetyl-CoA. In the mutant, citryl-CoA is only hydrolyzed, indicating a major defect in the condensation reaction. As monitored by the carbonyl carbon's chemical shift, the extent of oxaloacetate carbonyl polarization is decreased in all binary and ternary complexes. As indicated by the lack of rapid H320G--oxaloacetate catalysis of the exchange of the methyl protons of acetyl-CoA or the pro-S-methylene proton of propionyl-CoA, the activation of acetyl-CoA is also faulty. Reflecting this defect in acetyl-CoA activation, the carboxyl chemical shift of H320G-bound carboxymethyl-CoA (a transition-state analog of the neutral enol intermediate) fails to decrease on formation of the H3020G-oxaloacetate-carboxymethyl-CoA ternary complex. Progress curves and steady-state data with H320G using citryl-CoA as substrate show unusual properties: substrate inhibition and accelerating progress curves. Either one of two models with subunit cooperativity [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88; Koshland, D. E., Jr., Nemethy, G., & Filmer, D. (1966) Biochemistry 5, 365] quantitatively accounts for both the initial velocity data and the individual progress curves. The concentrations of all enzyme forms and complexes are assumed to rapidly reach their equilibrium values compared to the rate of substrate turnover. The native enzyme also behaves according to models for subunit cooperativity with citryl-CoA as substrate. However, the rates of formation/dissociation and reaction of complexes are kinetically significant. Comparisons of the values of kinetic constants between the native and mutants enzymes lead us to conclude that the mutant less readily undergoes a conformation change required for efficient activation of substrates. | Man WJ, Li Y, O'Connor CD, Wilton DC (1995)
The binding of propionyl-CoA and carboxymethyl-CoA to Escherichia coli citrate synthase.
Biochimica et biophysica acta 1250, 69-75 [PubMed:7612655]
[show Abstract]
The interaction of propionyl-CoA and acetyl-CoA with E. coli citrate synthase has been studied in order to gain insight into the structural requirements for substrate binding by this enzyme. In contrast to the enzyme from pig heart, the E. coli enzyme was unable to catalyse significant exchange of the methylene protons of propionyl-CoA while overall activity was very low with this enzyme. Carboxymethyl-CoA is a presumptive transition state analogue of acetyl-CoA using pig heart citrate synthase. The effect of carboxymethyl-CoA on both the native enzyme from E. coli and a catalytically active aspartate mutant (D362E) was investigated. Whereas the native enzyme was inhibited by carboxymethyl-CoA, the mutant enzyme (D362E) shows either no inhibition or minimal inhibition depending on the assay conditions. The binding of acetyl-CoA is not inhibited as a result of the mutation. The results with propionyl-CoA and carboxymethyl-CoA suggest that the active site of the E. coli enzyme is more restricted as compared with the enzyme from pig heart and, in the case of propionyl-CoA, this restriction prevents the formation of a catalytically productive enzyme-substrate complex. | Kurz LC, Shah S, Crane BR, Donald LJ, Duckworth HW, Drysdale GR (1992)
Proton uptake accompanies formation of the ternary complex of citrate synthase, oxaloacetate, and the transition-state analog inhibitor, carboxymethyl-CoA. Evidence that a neutral enol is the activated form of acetyl-CoA in the citrate synthase reaction.
Biochemistry 31, 7899-7907 [PubMed:1324722]
[show Abstract]
Citrate synthase complexes with the transition-state analog inhibitor, carboxymethyl-CoA (CM-CoA), are believed to mimic those with the activated form of acetyl-CoA. The X-ray structure [Karpusas, M., Branchaud, B., & Remington, S.J. (1990) Biochemistry 29, 2213] of the ternary complex of the enzyme, oxaloacetate, and CMCoA has been used as the basis for a proposal that a neutral enol of acetyl-CoA is that activated form. Since the inhibitor carboxyl has a pKa of 3.90, analogy with an enolic acetyl-CoA intermediate leads to the prediction that a proton should be taken up from solution upon formation of the analog complex so that the transition-state analog carboxyl is protonated when bound. We have obtained evidence in solution for this proposal by comparing the isoelectric points and the pH dependence of the dissociation constants of the ternary complexes of the pig heart enzyme with the neutral ground-state analog inhibitor, acetonyl-CoA (KCoA), and the anionic transition-state analog inhibitor (CMCoA) and by studying the NMR spectra of the transition-state analog complexes of allosteric (Escherichia coli) and nonallosteric (pig heart) enzymes. The pH dependence of the dissociation constant of the ground-state analog indicates no proton uptake, while that for the transition-state analog indicates that 0.55 +/- 0.04 proton is taken up when the analog binds to the citrate synthase-oxaloacetate binary complex. The overall charges of ternary complexes of the pig heart enzyme with the transition-state and ground-state analog inhibitors are the same, as monitored by their isoelectric points.(ABSTRACT TRUNCATED AT 250 WORDS) | Kurz LC, Drysdale GR, Riley MC, Evans CT, Srere PA (1992)
Catalytic strategy of citrate synthase: effects of amino acid changes in the acetyl-CoA binding site on transition-state analog inhibitor complexes.
Biochemistry 31, 7908-7914 [PubMed:1324723]
[show Abstract]
Acetyl-CoA enol has been proposed as an intermediate in the citrate synthase (CS) reaction with Asp375 acting as a base, removing a proton from the methyl carbon of acetyl-CoA, and His274 acting as an acid, donating a proton to the carbonyl [Karpusas, M., Branchaud, B., & Remington, S.J. (1990) Biochemistry 29, 2213]. CS-oxaloacetate (OAA) complexes with the transition-state analog inhibitor, carboxymethyl-CoA (CMCoA), mimic those with acetyl-CoA enol. Asp375 and His274 interact intimately with the carboxyl of the bound inhibitor. While enzymes in which these residues have been changed to other amino acids have very low catalytic activity, we find that they retain their ability to form complexes with substrates and the transition-state analog inhibitor. In comparison with the value of the chemical shift of the protonated CMCoA carboxyl in acidic aqueous solutions or its value in the wild-type ternary complex, the values in the Asp375 mutants are unusually low. Model studies suggest that these low values result from complete absence of one hydrogen bond partner for the Gly mutant and distortions in the active site hydrogen bond systems for the Glu mutant. The high affinity of Asp375Gly-OAA for CMCoA suggests that the unfavorable proton uptake required to stabilize the CMCoA-OAA ternary complex of the wild-type enzyme [Kurz, L.C., Shah, S., Crane, B.R., Donald, L.J., Duckworth, H.W., & Drysdale, G.R. (1992) Biochemistry (preceding paper in this issue)] is not required by this mutant; the needed proton is most likely provided by His274. This supports the proposed role of His274 as a general acid.(ABSTRACT TRUNCATED AT 250 WORDS) | Lill U, Kollmann-Koch A, Bibinger A, Eggerer H (1991)
Inhibitors of metabolic reactions. Scope and limitation of acyl-CoA-analogue CoA-thioethers.
European journal of biochemistry 198, 767-773 [PubMed:1675605]
[show Abstract]
Substrate and intermediate analogue inhibitors of enzymes were prepared in which the thioester oxygen of acyl-CoA substrates is replaced by hydrogen with formation of CoA-thioethers. Experiments performed with ATP citrate lyase and S-(3,4-dicarboxy-3-hydroxybutyl)-CoA are consistent with citryl-CoA but not with citryl-enzyme being the direct precursor of the products acetyl-CoA and oxaloacetate. Consistent with these results, a previously described isotopic exchange between acetyl-CoA and [3H]CoASH, indicating the formation of an acetyl-enzyme in the reaction pathway, could not be confirmed. Substrate analogue CoA-thioethers of malate synthase are inhibitors endowed with the affinity of the substrates. Acetyl carboxylase and fatty acid synthetase are not inhibited by the substrate analogue S-ethyl-CoA; S-carboxyethyl-CoA, which could substitute for malonyl-CoA, is likewise not inhibitory. An explanation is proposed. Previously suggested roles of S-carboxymethyl-CoA, an acetyl-CoA-related inhibitor of citrate synthase, are discussed in the light of new experimental data. S-Acetyl, S-propionyl and S-carboxymethyl derivatives of 1,N6-etheno-CoA loose the high affinity of their CoA-counterparts to citrate synthase, probably because the ethylene group prevents proper binding to the enzyme. | Handford PA, Ner SS, Bloxham DP, Wilton DC (1988)
Site-directed mutagenesis of citrate synthase; the role of the active-site aspartate in the binding of acetyl-CoA but not oxaloacetate.
Biochimica et biophysica acta 953, 232-240 [PubMed:3281713]
[show Abstract]
Asp-362, a potential key catalytic residue of Escherichia coli citrate synthase (citrate oxaloacetate-lyase [pro-3S)-CH2COO- ----acetyl-CoA), EC 4.1.3.7) has been converted to Gly-362 by oligonucleotide-directed mutagenesis. The mutant gene was completely sequenced, using a series of synthetic oligodeoxynucleotides spanning the structural gene to confirm that no additional mutations had occurred during genetic manipulation. The mutant gene was expressed in M13 bacteriophage and produced a protein which migrated in an identical manner to wild-type E. coli citrate synthase on SDS-polyacrylamide gels and which cross-reacted with E. coli citrate synthase antiserum. The mutant gene was subsequently recloned into pBR322 for large scale purification of the protein, and the resulting plasmid, pCS31, used to transform the citrate synthase deletion strain, W620. The mutant enzyme purified in an analogous manner to wild-type E. coli citrate synthase and expressed less than 2% of wild-type enzyme activity. The activity of the partial reactions catalysed by citrate synthase was similarly affected suggesting that this residual activity may be due to contaminating wild-type enzyme activity. The mutant citrate synthase retains a high-affinity NADH-binding site consistent with the protein preserving its overall structural integrity. Oxaloacetate binding to the protein is unaffected by the Asp-362 to Gly-362 mutation. Binding of the acetyl-CoA analogue, carboxymethyl-CoA, could not be detected in the mutant protein indicating that the lack of catalytic competence is due primarily to the inability of the protein to bind the second substrate, acetyl-CoA. | Kurz LC, Drysdale GR (1987)
Evidence from Fourier transform infrared spectroscopy for polarization of the carbonyl of oxaloacetate in the active site of citrate synthase.
Biochemistry 26, 2623-2627 [PubMed:3607038]
[show Abstract]
The infrared spectrum of oxaloacetate bound in the active site of citrate synthase has been measured in the binary complex and in the ternary complex with the acetyl coenzyme A (CoA) enolate analogue carboxymethyl-CoA. The carbonyl stretching frequency of oxaloacetate in binary and ternary complexes is found at 1697 cm-1, a shift of 21 cm-1 to lower frequency relative to that of the free ligand. The line widths of the carbonyl absorption in enzyme complexes differ from that of the free ligand, decreasing from a value of 20 cm-1 for the free ligand to 10 cm-1 in the binary complex and 7 cm-1 in the ternary complex with carboxymethyl-CoA. The integrated absorbance of the carbonyl absorption in these enzyme complexes is significantly increased over that of the free ligand at the same concentration, increasing approximately 2-fold in the binary complex and approximately 3-fold in the ternary complex. These results indicate strong polarization of the carbonyl bond in the enzyme-substrate complexes and suggest that ground-state destabilization is a major catalytic strategy of citrate synthase. | Kurz LC, Ackerman JJ, Drysdale GR (1985)
Evidence from 13C NMR for polarization of the carbonyl of oxaloacetate in the active site of citrate synthase.
Biochemistry 24, 452-457 [PubMed:3978085]
[show Abstract]
The carbon-13 NMR spectrum of oxaloacetate bound in the active site of citrate synthase has been obtained at 90.56 MHz. In the binary complex with enzyme, the positions of the resonances of oxaloacetate are shifted relative to those of the free ligand as follows: C-1 (carboxylate), -2.5 ppm; C-2 (carbonyl), +4.3 ppm; C-3 (methylene), -0.6 ppm; C-4 (carboxylate), +1.3 ppm. The change observed in the carbonyl chemical shift is successively increased in ternary complexes with the product [coenzyme A (CoA)], a substrate analogue (S-acetonyl-CoA), and an acetyl-CoA enolate analogue (carboxymethyl-CoA), reaching a value of +6.8 ppm from the free carbonyl resonance. Binary complexes are in intermediate to fast exchange on the NMR time scale with free oxaloacetate; ternary complexes are in slow exchange. Line widths of the methylene resonance in the ternary complexes suggest complete immobilization of oxaloacetate in the active site. Analysis of line widths in the binary complex suggests the existence of a dynamic equilibrium between two or more forms of bound oxaloacetate, primarily involving C-4. The changes in chemical shifts of the carbonyl carbon indicate strong polarization of the carbonyl bond or protonation of the carbonyl oxygen. Some of this carbonyl polarization occurs even in the binary complex. Development of positive charge on the carbonyl carbon enhances reactivity toward condensation with the carbanion/enolate of acetyl-CoA in the mechanism which has been postulated for this enzyme. The very large change in the chemical shift of the reacting carbonyl in the presence of an analogue of the enolate of acetyl-CoA supports this interpretation. | Bayer E, Bauer B, Eggerer H (1981)
Evidence from inhibitor studies for conformational changes of citrate synthase.
European journal of biochemistry 120, 155-160 [PubMed:7308213]
[show Abstract]
1. Substrate analogue CoA derivatives were applied as inhibitors of citrate synthase. Substitution of the acyl-CoA oxygen next to sulfur by hydrogen was without marked influence on the affinity. 2. Carboxymethyl-CoA, a structural analogue of enolic acetyl-CoA, was characterized as a transition state analogue by an affinity 100-fold higher than that of acetyl-CoA. Ks of the binary inhibitor-enzyme complex was high (230 microM) but that of the ternary inhibitor-oxaloacetate-enzyme complex was 0.07 microM. Both enzyme subunits bound the inhibitor independently, also in the presence of oxaloacetate. 3. (3R,S)-3,4-Dicarboxy-3-hydroxybutyl-CoA, an analogue of citryl-CoA, inhibited the overall reaction noncompetitively against acetyl-CoA and against oxaloacetate; it was a competitive inhibitor against the hydrolysis and cleavage reactions of (3S)-citryl-CoA. Kinetic data suggest that this inhibitor represents an intermediate analogue. 4. The results given above indicate conformational changes of the synthase during the catalytic cycle. In the proposed mechanism the free enzyme represents a hydrolase which in the presence of oxaloacetate, by a well-known conformational change, is converted into a ligase. If both substrates are present, the ligase is reconverted into the hydrolase upon formation of the intermediate, (3S)-citryl-CoA. |
|
