Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.3.1 (citrate synthase)
4,488 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The crystal structure of pig heart citrate synthase was analyzed at 0.35-nm resolution. Chain tracing was possible and an initial molecular model constructed. The dimensions of the dimer molecule (located on a crystallographic diad) are 7.5 x 6.0 x 9.0 nm. The chain folding is characterized by the predominance of helices and the absence of sheet structure. The electron density accounts for 355 residues per monomer, so that about 80 residues must be disordered in the crystal. The disordered segment in probably N-terminal. The ordered part consists of two closely associated domains, a large domain with 300 residues and a C-terminal domain of 55 residues consisting of 3(anti)parallel helices. The large domain is built from 12 helical segments, some of which are buried in the interior of the molecule. Inhibitor binding studies with citrate and CoA revealed citrate binding sites but showed no electron density for CoA. It is suggested that CoA binds to the disordered, flexible N-terminal domain. Experiments of limited proteolysis with trypsin showed that under conditions a segment of Mr 9000 is cleaved off selectively. The remaining 35 000-Mr part is dimeric.
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PMID:Crystal structure analysis of the tetragonal crystal form are preliminary molecular model of pig-heart citrate synthase. 43 30

Two methods are developed for the theoretical determination of a conformational path between two well-documented forms, a closed form and the open form [Remington et al. (1982) J. Mol. Biol. 158, 111-152] of pig heart citrate synthase, a dimeric enzyme of 2 x 437 residues. The first method uses the minimization of the sum of the potential energies at a set of equidistant points, according to Elber and Karplus [(1987) Chem. Phys. Lett. 139, 375-380]. The initialization of the algorithm is modified to account for large-angle rotations of many groups by performing the interpolations in the space of internal polar coordinates of a set of generalized Jacobi vectors earlier introduced by Durup [(1991) J. Phys. Chem. 95, 1817-1829] and by carefully testing all choices of directions of rotation for determining the initialized midpoint between the known forms. The path includes intermediate points, created by successive splittings of each interval into two equal parts, with a partial energy minimization performed after each splitting. The minimization encounters the well-known local-minima problem, which here is handled by low-temperature molecular dynamics annealing. It is shown that the best ratio of potential energy decrease to rms deviation is achieved by running the dynamics at 50 K, as compared to 100 K and above. The main character of the path obtained is the occurrence of strong to-and-fro variations of some dihedral angles at specific stages along the path. The second method, which we name directed dynamics, uses only low-temperature molecular dynamics simulations by starting trajectories from each of the two known forms with initial velocities directed toward the other one. The procedure is iterated by restarting trajectory pairs after the points of closest approach of the preceding pair. The two half-paths thus built eventually meet after 70 iterations. This method provides a second path with strong similarities, as well as some differences, with respect to the path obtained by the first method.
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PMID:Theoretical determination of conformational paths in citrate synthase. 151 47

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.
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PMID:Conversion, by limited proteolysis, of an archaebacterial citrate synthase into essentially a citryl-CoA hydrolase. 152 37

The molecular chaperone GroE facilitates correct protein folding in vivo and in vitro. The mode of action of GroE was investigated by using refolding of citrate synthase as a model system. In vitro denaturation of this dimeric protein is almost irreversible, since the refolding polypeptide chains aggregate rapidly, as shown directly by a strong, concentration-dependent increase in light scattering. The yields of reactivated citrate synthase were strongly increased upon addition of GroE and MgATP. GroE inhibits aggregation reactions that compete with correct protein folding, as indicated by specific suppression of light scattering. GroEL rapidly forms a complex with unfolded or partially folded citrate synthase molecules. In this complex the refolding protein is protected from aggregation. Addition of GroES and ATP hydrolysis is required to release the polypeptide chain bound to GroEL and to allow further folding to its final, active state.
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PMID:GroE facilitates refolding of citrate synthase by suppressing aggregation. 167 55

Citrate synthase (EC 4.1.3.7), which is present in all living organisms as a key enzyme in aerobic energy metabolism, is one of the most highly phylogenetically conserved enzymes known in terms of its primary and active site structure. However, in terms of other parameters such as in vitro stability, tolerance to changes in pH, degree of self-polymerization, etc., citrate synthases from different sources are markedly different. These divergences can be observed even between isoforms of the enzyme within the same species. Data documenting these diversities suggest that a high degree of difference in tertiary structures may occur. Therefore, the surface profiles of citrate synthase enzymes from yeast, pig, rat, tomato and Escherichia coli were investigated with immunological methods using monoclonal antibody families generated against either pig citrate synthase (alpha-PCS) or yeast citrate synthase-2 (alpha-YCS-2). A high degree of homology of enzyme epitopes was detected on the mitochondrial citrate synthases originating from yeast, tomato, pig and rat cells. Major differences were found between the hexameric citrate synthase originating from E. coli compared with those dimeric forms prepared from eukaryotic cells. Only modest similarities were detected between the highly homologous peroxisomal and mitochondrial yeast citrate synthases. Furthermore, a point mutation of one of the catalytic residues (H274R on recombinant pig and H313R on yeast enzyme) of mitochondrial citrate synthase (CS-1) resulted in a significant increase in immunological similarity with the peroxisomal isoenzyme (CS-2). These findings are discussed in terms of the possible mechanism of evolution of CS-2 in yeast.
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PMID:Immunological mapping of fine molecular surface structures of citrate synthase enzymes from different cell types. 181 Mar 49

The unfolding of the dimeric enzyme citrate synthase from pig heart in solutions of guanidinium chloride (GdnHCl) was studied. Data from fluorescence, circular dichroism (CD) and thiol group reactivity studies indicated that the enzyme was almost completely unfolded at GdnHCl concentrations greater than or equal to 4 M. On dilution of GdnHCl, essentially no reactivation of the enzyme occurred. The implications of this finding for the process of folding and assembly in vivo of this and other mitochondrial enzymes are discussed. Exposure of the enzyme to high pH (9-10) led to only a small loss of secondary structure and partial reactivation could be observed on readjustment of the pH to 8.0.
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PMID:The unfolding and attempted refolding of citrate synthase from pig heart. 231 Jul 49

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.
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PMID:Models of proteolysis of oligomeric enzymes and their applications to the trypsinolysis of citrate synthases. 314 Aug 3

Porcine heart citrate synthase, a dimeric protein of Mr = 100,000 composed of two identical subunits, is shown to undergo a monomer-dimer equilibrium. The extent of dimerization is found to be dependent on the concentration of citrate synthase, pH, ionic strength, and the specific buffer system employed. Oxaloacetate and citrate, substrates for the forward and reverse reaction catalyzed by citrate synthase, affect dimerization at concentrations of the protein which exists as monomer in their absence. The dissociation of citrate synthase dimers has been demonstrated utilizing the techniques of gel permeation chromatography, fluorescence polarization, fluorescence energy transfer, and heat denaturation. Earlier studies of citrate synthase quarternary structure found the protein to be nondissociable except under denaturing conditions or extensive modification; however, most former studies were performed at relatively high protein concentration, ionic strength, and pH, conditions which stabilize the dimer. In light of recent evidence derived from x-ray crystallographic studies showing amino acid residues from one subunit contributing to the citrate and CoA binding sites of the other, the dissociation into monomers would be expected to have profound effects on citrate synthase activity and regulation, as well as overall tricarboxylic acid cycle activity.
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PMID:Subunit equilibria of porcine heart citrate synthase. Effects of enzyme concentration, pH, and substrates. 394 36

Detailed evidence for the amino acid sequence of allosteric citrate synthase from Escherichia coli is presented. The evidence confirms all but 11 of the residues inferred from the sequence of the gene as reported previously [Ner, S. S., Bhayana, V., Bell, A. W., Giles, I. G., Duckworth, H. W., & Bloxham, D. P. (1983) Biochemistry 22, 5243]; no information has been obtained about 10 of these (residues 101-108 and 217-218), and we find aspartic acid rather than asparagine at position 10. Substantial regions of sequence homology are noted between the E. coli enzyme and citrate synthase from pig heart, especially near residues thought to be involved in the active site. Deletions or insertions must be assumed in a number of places in order to maximize homology. Either of two lysines, at positions 355 and 356, could be formally homologous to the trimethyllysine of pig heart enzyme, but neither of these is methylated. It appears that E. coli and pig heart citrate synthases are formed of basically similar subunits but that considerable differences exist, which must explain why the E. coli enzyme is hexameric and allosterically inhibited by NADH, while the pig heart enzyme is dimeric and insensitive to that nucleotide.
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PMID:Amino acid sequence of Escherichia coli citrate synthase. 638 May 76

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.
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PMID:Citrate synthase from a Gram-positive bacterium. Purification and characterization of the Bacillus megaterium enzyme. 641 81


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