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)

Chicken liver fatty acid synthase is inhibited by the thiol-modifying reagents 5,5'-dithiobis-(2-nitrobenzoic acid) and iodoacetamide. Total inactivation of the activity for fatty acid synthesis requires the modification of about 8 of the nearly 50 freely accessible thiol groups per molecule. The differential binding of iodo[14C]acetamide to phenylmethylsulphonyl fluoride-modified enzyme in the absence and in the presence of excess acetyl-CoA shows complete modification of one cysteine-SH site of the condensing enzyme and partial modification of the pantetheine-SH site for a total of approx. 1.4 mol of iodoacetamide bound per mol of enzyme. The reaction of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) generates disulphide cross-links for each molecule of the reagent added, but 95% of these cross-links are intrasubunit. Both the iodoacetamide- and 5,5'-dithiobis-(2-nitrobenzoic acid)-modified species catalyse all the component partial reactions of fatty acid synthesis except the condensation reaction. The results obtained with iodoacetamide show that in the dimeric fatty acid synthase modification of one cysteine-SH condensing site and/or one pantetheine-SH site per dimer is sufficient to affect inhibition of condensing activity and the activity for fatty acid synthesis, and are in accord with a recently proposed model for the mechanism of action of animal fatty acid synthases [Kumar (1982) J. Theor. Biol. 95, 263-283].
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PMID:Inhibition of the condensing component of chicken liver fatty acid synthase by iodoacetamide and 5,5'-dithiobis-(2-nitrobenzoic acid). 666 Nov 83

The binding of two similar spin-labeled fatty acyl-CoA analogues, one short chain, 6-doxyloctanoyl-CoA (S-(2-(5-carboxybutyl)-2-ethyl-4, 4-dimethyl-3-oxazolidinyl-N-oxyl)-CoA) and one long chain, 6-doxylstearoyl-CoA (S-(2-(5-carboxybutyl)-2-dodecyl-4, 4-dimethyl-3-oxazolidinyl-N-oxyl)-CoA) to pig heart citrate synthase (citrate oxaloacetate-lyase (pro-3S-CH2COO- leads to acetyl-CoA) EC 4.1.3.7) has been compared. The binding of the short chain analogue could be satisfactorily fit by a classical treatment (independent, noninteracting sites) with well defined stoichiometry: 2 mol of spin label bound per mol of dimeric enzyme. Binding of the long chain analogue was complex and in excess of 2 mol/dimer. Competitive binding experiments using either analogue in the presence of various nucleotides and substrates revealed differences in the binding of the long and short chain analogues. These additional studies, together with kinetic measurements, implied isosteric binding of acyl-CoA, ATP, NADPH, NADH, NADP+, acetyl-CoA, and partial isosteric binding of the long chain acyl-CoA. Binding of NADPH and NADP+ to the same form of the enzyme, perhaps through overlapping sites, was kinetically verified even though these nucleotides had differing effects on the binding of the spin-labeled analogues. Oxalacetate was shown to decrease the binding of the long chain analogue but to have no effect on the binding of the short chain. This result was supported by kinetic measurements. The competitive binding experiments with the long chain analogue suggested that its complex isotherm resulted from binding in two classes of sites, i.e. two cooperative nucleotide sites and other sites. An empirical mathematical model employing this rationale provided a satisfactory fit for the binding of fatty acyl-CoA to citrate synthase. A spin-labeled fatty acid which was not bound by the native enzyme was appreciably bound in the presence of additional palmitoyl-CoA. This binding might be identified with one of the two sets of binding sites proposed in the model. These and previous results on acyl-CoA binding were correlated with the properties of the CoA binding site defined crystallographically (Remington, S., Wiegand, G., and Huber, R. (1982) J. Mol. Biol. 158, 111-152).
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PMID:Regulation of enzymes by fatty acyl coenzyme A. Interactions of short and long chain spin-labeled acyl-CoA with the acetyl-CoA site on pig heart citrate synthase. 669 13

The crystal structure of the complex of pig heart citrate synthase and oxaloacetate in the presence of the potent inhibitor S-acetonyl coenzyme A has been determined at a nominal resolution of 2.9 A by Patterson search techniques and refined by restrained crystallographic refinement. The complex crystallizes in the presence of polyvinylpyrrolidone in space group P4(3)2(1)2 with a = 101.5 A and c = 224.6 A, with one dimeric molecule of molecular weight 100,000 in the asymmetric unit. The crystallographic R factor is 0.194 for the 14,332 unique reflections between 6.0 and 2.9 A resolution. The structures of two forms of citrate synthase in the presence and absence of product molecules have been determined recently and shown to differ in the relative arrangement of the large and small domains ("closed" and "open" forms). The third crystal form described here is also closed, but there is substantial rearrangement within the small domain relative to either of the other crystal forms. We conclude that this is a third structural state of the enzyme, and catalytic activity of the enzyme depends on structural changes during the course of the reaction affecting domain conformation also. The three structures are compared, and it is shown that the large domain is considerably more rigid than the small domain. The conformation of the small domain adapts to the ligand. The inhibitor, and the "coenzyme-A-binding segment" of the enzyme are disordered. No electron density is observed for the inhibitor, and only weak density for the coenzyme-A-binding segment. Electron density for oxaloacetate is well defined. It binds in a very similar manner to citrate.
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PMID:Crystal structure analysis and molecular model of a complex of citrate synthase with oxaloacetate and S-acetonyl-coenzyme A. 671 77

At normal temperatures, Hsp90 is one of the most abundant proteins in the cytosol of various eucaryotic cells. Upon heat shock, the level of Hsp90 is increased even more, suggesting that it is important for helping cells to survive under these conditions. However, studies so far have been almost exclusively concerned with the function of Hsp90 under non-stress conditions, and therefore only little is known about the role of Hsp90 during heat shock. As a model for heat shock in vitro, we have monitored the inactivation and subsequent aggregation of dimeric citrate synthase (CS) at elevated temperatures. Hsp90 effectively "stabilized" CS under conditions where the enzyme is normally inactivated and finally aggregates very rapidly. A kinetic dissection of the unfolding pathway of CS succeeded in revealing two intermediates which form and subsequently undergo irreversible aggregation reactions. Hsp90 apparently interacts transiently with these highly structured early unfolding intermediates. Binding and subsequent release of the intermediates favorably influences the kinetic partitioning between two competing processes, the further unfolding of CS and the productive refolding to the native state. As a consequence, CS is effectively stabilized in the presence of Hsp90. The significance of this interaction is especially evident in the suppression of aggregation, the major end result of thermal unfolding events in vivo and in vitro. These effects, which are ATP-independent, seem to be a general function of members of the Hsp90 family, since yeast and bovine Hsp90 as well as the Hsp90 homologue from Escherichia coli gave similar results. It seems likely that this function also reflects the role of Hsp90 under heat shock conditions in vivo. We therefore propose that members of the Hsp90 family convey thermotolerance by transiently binding to highly structured early unfolding intermediates, thereby preventing their irreversible aggregation and stabilizing the active species.
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PMID:Transient interaction of Hsp90 with early unfolding intermediates of citrate synthase. Implications for heat shock in vivo. 770 69

Escherichia coli possesses a hexameric citrate synthase that exhibits allosteric kinetics and regulatory sensitivity, and for which the gene (gltA) has previously been cloned and sequenced. A citrate-synthase-deficient strain of E. coli (K114) has been mutated to generate a revertant (K114r4) that produces a dimeric citrate synthase with altered kinetic and regulatory properties. On cloning and sequencing the gltA gene from both K114 and K114r4, a single mutation was found that caused the replacement of Asp362 with Asn. Asp362 has been previously shown to be a catalytically essential residue in E. coli citrate synthase, and we demonstrate that the hexameric enzyme produced on expression of the gltA gene from K114 and K114r4 is inactive. The dimeric citrate synthase from K114r4 has been purified and shown to be immunologically distinct from the wild-type hexameric enzyme. Determination of its N-terminal amino acid sequence demonstrates that the mutant citrate synthase is encoded by a gene distinct from the E. coli gltA gene. The N-terminal sequence is compared with those of other eukaryotic, eubacterial and archaebacterial citrate synthases.
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PMID:Does Escherichia coli possess a second citrate synthase gene? 850 9

A normal mode analysis of the closed form of dimeric citrate synthase has been performed. The largest-amplitude collective motion predicted by this method compares well with the crystallographically observed hinge-bending motion. Such a result supports those obtained previously in the case of hinge-bending motions of smaller systems, such as lysozyme or hexokinase. Taken together, all these results suggest that low-frequency normal modes may become useful for determining a first approximation of the conformational path between the closed and open forms of these proteins.
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PMID:Hinge-bending motion in citrate synthase arising from normal mode calculations. 874 51

The crystal structure of the closed form of citrate synthase, with citrate and CoA bound, from the hyperthermophilic Archaeon Pyrococcus furiosus has been determined to 1.9 A. This has allowed direct structural comparisons between the same enzyme from organisms growing optimally at 37 degrees C (pig), 55 degrees C (Thermoplasma acidophilum) and now 100 degrees C (Pyrococcus furiosus). The three enzymes are homodimers and share a similar overall fold, with the dimer interface comprising primarily an eight alpha-helical sandwich of four antiparallel pairs of helices. The active sites show similar modes of substrate binding; moreover, the structural equivalence of the amino acid residues implicated in catalysis implies that the mechanism proceeds via the same acid-base catalytic process. Given the overall structural and mechanistic similarities, it has been possible to make detailed structural comparisons between the three citrate synthases, and a number of differences can be identified in passing from the mesophilic to thermophilic to hyperthermophilic citrate synthases. The most significant of these are an increased compactness of the enzyme, a more intimate association of the subunits, an increase in intersubunit ion pairs, and a reduction in thermolabile residues. Compactness is achieved by the shortening of a number of loops, an increase in the number of atoms buried from solvent, an optimized packing of side chains in the interior, and an absence of cavities. The intimate subunit association in the dimeric P. furiosus enzyme is achieved by greater complementarity of the monomers and by the C-terminal region of each monomer folding over the surface of the other monomer, in contrast to the pig enzyme where the C-terminus has a very different fold. The increased number of intersubunit ion pairs is accompanied by an increase in the number involved in networks. Interestingly, all loop regions in the P. furiosus enzyme either are shorter or contain additional ion pairs compared with the pig enzyme. The possible relevance of these structural features to enzyme hyperthermostability is discussed.
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PMID:The crystal structure of citrate synthase from the hyperthermophilic archaeon pyrococcus furiosus at 1.9 A resolution,. 925 93

The murine small heat shock protein Hsp25 carries a single cysteine residue in position 141 of its amino acid sequence. Interestingly, Hsp25 can exist within the cell as covalently bound dimer which is linked by an intermolecular disulfide bond between two monomers. Oxidative stress caused by treatment of the cells with diamide, arsenite, or hydrogen peroxide leads to an increase in Hsp25-dimerisation which can be blocked by simultaneous treatment with reducing agents. Recombinant Hsp25 was prepared in an oxidized dimeric (oxHsp25) and reduced monomeric (redHsp25) from. The two species were compared with regard to secondary structure, stability, oligomerization properties and their chaperone activity. It is demonstrated by CD measurements in the far UV region that there are no significant differences in the secondary structure and temperature- or pH-stability of oxHsp25 and redHsp25. However, according to CD measurements in the near UV region an increase in the asymmetry of the microenvironment of aromatic residues in oxHsp25 is observed. Furthermore, an increase in stability of the hydrophobic environment of the tryptophan residues mainly located in the N-terminal domain of the protein against urea denaturation is detected in oxHsp25. Both reduced and oxidized Hsp25 from oligomeric complexes of similar size and stability against detergents and both species prevent thermal aggregation of citrate synthase and assist significantly in oxaloacetic acid-induced refolding of the enzyme. Hence, the overall secondary structure, the degree of oligomerization and the chaperone activity of Hsp25 seem independent of the formation of the intermolecular disulfide bond and only the stability of the hydrophobic N-terminal part of the molecule is influenced by formation of this bound. The obtained data do not exclude the possible involvement of dimerization of this protein in other cellular functions, e.g. in intracellular sulfhydryl-buffering or in the protection of actin filaments from fragmentation upon oxidative stress.
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PMID:The effect of the intersubunit disulfide bond on the structural and functional properties of the small heat shock protein Hsp25. 965 71

The prokaryotic molecular chaperone GroE is increasingly expressed under heat shock conditions. GroE protects cells by preventing the irreversible aggregation of thermally unfolding proteins. Here, the interaction of GroE with thermally unfolding citrate synthase (CS) was dissected into several steps that occur before irreversible aggregation, and the conformational states of the unfolding protein recognized by GroEL were determined. The kinetic analysis of CS unfolding revealed the formation of inactive dimeric and monomeric intermediates. GroEL binds both intermediates without affecting the unfolding pathway. Furthermore, the dimeric intermediates are not protected against dissociation in the presence of GroEL. Monomeric CS is stably associated with GroEL, thus preventing further irreversible unfolding steps and subsequent aggregation. During refolding, monomeric CS is encapsulated inside the cavity of GroEL. GroES complexes. Taken together our results suggest that for protection of cells against heat stress both the ability of GroEL to interact with a large variety of nonnative conformations of proteins and the active, GroES-dependent refolding of highly unfolded species are important.
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PMID:GroEL traps dimeric and monomeric unfolding intermediates of citrate synthase. 983 3

Beta-cyclodextrin (CD) dimers (n = 11) were synthesized and tested against eight enzymes, seven of which were dimeric or tetrameric, for inhibitor activity. Initial screening showed that only L-lactate dehydrogenase and citrate synthase were inhibited but only by two specific CD dimers in which two beta-CDs were linked on the secondary face by a pyridine-2,6-dicarboxylic group. Further investigation suggested that these CD dimers inhibit the activity of L-lactate dehydrogenase and citrate synthase at least in part by disruption of protein-protein aggregation.
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PMID:Selective disruption of protein aggregation by cyclodextrin dimers. 1080 68


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