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

---

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

An alpha-fluoro acid analog and an alpha-fluoro amide analog of acetyl-CoA have been synthesized. The ternary complexes of these inhibitors with oxaloacetate and citrate synthase have been crystallized and their structures analyzed at 1.7 A resolution. The structures are similar to those reported for the corresponding non-fluorinated analogs (Usher et al., 1994), with all forming unusually short hydrogen bonds to Asp 375. The alpha-fluoro amide analog binds with an affinity 1.5-fold lower than that of a previously described amide analog lacking the alpha-fluoro group. The alpha-fluoro acid analog binds with a 50-fold decreased affinity relative to the corresponding unfluorinated analog. The binding affinities are consistent with increased strengths of hydrogen bonds to Asp 375 with closer matching of pKa values between hydrogen bond donors and acceptors. The results do not support any direct correlation between hydrogen bond strength and hydrogen bond length in enzyme-inhibitor complexes.
...
PMID:alpha-Fluoro acid and alpha-fluoro amide analogs of acetyl-CoA as inhibitors of citrate synthase: effect of pKa matching on binding affinity and hydrogen bond length. 749 47

The effect of dietary vitamin E supplementation upon macrophage metabolism and function was examined in aged rats fed a balanced or a polyunsaturated-rich diet. The following parameters were studied: number of cells in the intraperitoneal cavity, maximal activity of hexokinase, citrate synthase, glucose-6-phosphate dehydrogenase, glutathione peroxidase and phosphate-dependent glutaminase. The consumption of glucose and the production of lactate, hydrogen peroxide and thiobarbituric reactive substances were measured in control ONCO-BCG injected rats. The results indicated that vitamin E has no significant effect on the values of the parameters studied in the macrophages of rats fed a balanced diet both for 3 (mature) or 17 months (aged). This antioxidant did not provoke any response on the changes caused by ageing the animals. However, several of the metabolic and functional alterations in macrophage induced by the polyunsaturated-rich diets were reversed by the inclusion of vitamin E in the diet. These changes were associated with macrophage migration capacity, citrate synthase and glucose-6-phosphate dehydrogenase activities and the content of lipid peroxides. The findings suggest that vitamin E has a beneficial effect for macrophage metabolism and function, but the effects are confined to particular circumstances.
...
PMID:Effect of dietary vitamin E supplementation on macrophage metabolism during ageing. Study in rats fed fat-rich diets during ageing. 784 17

Two extremely potent inhibitors of citrate synthase, carboxyl and primary amide analogues of acetyl coenzyme A, have been synthesized. The ternary complexes of these inhibitors with oxaloacetate and citrate synthase have been crystallized and their structures analyzed at 1.70- and 1.65-A resolution, respectively. The inhibitors have dissociation constants in the nanomolar range, with the carboxyl analogue binding more tightly (Ki = 1.6 nM at pH 6.0) than the amide analogue (28 nM), despite the unfavorable requirement for proton uptake by the former. The carboxyl group forms a shorter hydrogen bond with the catalytic Asp 375 (distance < 2.4 A) than does the amide group (distance approximately 2.5 A). Particularly with the carboxylate inhibitor, the very short hydrogen bond distances measured suggest a low barrier or short strong hydrogen bond. However, the binding constants differ by only a factor of 20 at pH 6.0, corresponding to an increase in binding energy for the carboxyl analogue on the enzyme of about 2 kcal/mol more than the amide analogue, much less than has been proposed for short strong hydrogen bonds based on gas phase measurements [> 20 kcal/mol (Gerlt & Gassman, 1993a,b)]. The inhibitor complexes support proposals that Asp 375 and His 274 work in concert to form an enolized form of acetyl-coenzyme A as the first step in the reaction.
...
PMID:A very short hydrogen bond provides only moderate stabilization of an enzyme-inhibitor complex of citrate synthase. 801 40

In vitro specific transcription by the Rickettsia prowazekii RNA polymerase was investigated. The purified rickettsial RNA polymerase, in striking contrast to that of Escherichia coli, could specifically transcribe two R. prowazekii genes (ATP/ADP translocase and citrate synthase genes) and one E. coli gene (RNA-I) on negative supercoiled plasmids but not the same genes on linear plasmids. Following the specific binding of the rickettsial RNA polymerase to the translocase gene promoter on a linear plasmid, there was no detectable open complex formation. Both the E. coli and the R. prowazekii RNA polymerases worked well when poly(dA-dT).poly(dA-dT) or poly(dI-dC).poly-(dI-dC) was used as template for generalized transcription. However, the rickettsial RNA polymerase, in contrast to the E. coli enzyme, had little activity on poly(dG-dC).poly(dG-dC), a template with a larger number of hydrogen bonds. These data indicate that the rickettsial RNA polymerase is weak, at least relative to E. coli, in the function required for the opening of DNA duplex. It appears that this operation in R. prowazekii is aided by the negative supercoiling and the high 72% AT composition of the rickettsial genome.
...
PMID:Characterization of the DNA-melting function of the Rickettsia prowazekii RNA polymerase. 844 Jun 83

We examined the catalytic efficiency of 18 pig citrate synthase mutants. The residues mutated were selected according to two criteria: the conservation of that residue in all known citrate synthase sequences, and the importance of that residue in substrate-amino acid interactions suggested by the extensive crystal structure information on the enzyme and its complexes. Several changes were made at certain residues to probe the effects of size, hydrogen bonding, and charge on the kinetics of the enzyme. The mutations, as expected, affected the kcats and Kms for OAA and acetyl-CoA to varying degrees. The catalytic efficiency of each of the mutants was determined by the kcat/Km for the individual substrates, OAA and acetyl-CoA. All mutations affected kcat. There was only one mutant, Asp327 Asn, in which the Kms primarily were affected. Most mutations affected both kcat and Km and included the following: His274Gly, His274Arg, Asp375Gly, Asp375Asn, Asp375Glu, Asp375Gln, His320Gly, His320Gln, His320Asn, His320Arg, Arg401His, Gly275Val, and Gly275Ala. The mutations, Arg401Gly, Arg401Lys, His235Gln, and Asn242Glu, had smaller effects on kcat and Km. The CS mutant Arg401Lys exhibited a modestly improved kcat/Km for both substrates compared to the nonmutant enzyme. X-ray crystallographic studies at 2.7 A resolution of one of the mutants, His274Gly, have been undertaken. The mutant enzyme crystallizes in an "open" conformation essentially isomorphous to wild type. The refined model has good geometry and a crystallographic R factor of 0.187 for 11 441 reflections observed between 6.0 and 2.7 A resolution. The refined model revealed a localized relaxation of the structure to relieve strain imposed by a high-energy main and side chain conformation of His274 in the nonmutant, but otherwise the mutation does not result in major structural alterations. Preliminary electrostatic calculations provide support for the concept that the transition state in the rate-limiting step of the citrate synthase catalyzed reaction may be an "enolized" version of acetyl-CoA that is neither neutral nor fully negatively charged and that a possible role for the catalytically essential His274 is to stabilize this by charge delocalization mediated by a hydrogen bond. These results provide the basis for further studies of the effects of these changes on the several reactive intermediates, activated substrates, and transition states which may occur along the reaction coordinate for this type of Claisen enzyme.
...
PMID:Active site mutants of pig citrate synthase: effects of mutations on the enzyme catalytic and structural properties. 871 55

Citrate synthase forms citrate by deprotonation of acetyl-CoA followed by nucleophilic attack of this substrate on oxaloacetate, and subsequent hydrolysis. The rapid reaction rate is puzzling because of the instability of the postulated nucleophilic intermediate, the enolate of acetyl-CoA. As alternatives, the enol of acetyl-CoA, or an enolic intermediate sharing a proton with His-274 in a "low-barrier" hydrogen bond have been suggested. Similar problems of intermediate instability have been noted in other enzymic carbon acid deprotonation reactions. Quantum mechanical/molecular mechanical calculations of the pathway of acetyl-CoA enolization within citrate synthase support the identification of Asp-375 as the catalytic base. His-274, the proposed general acid, is found to be neutral. The acetyl-CoA enolate is more stable at the active site than the enol, and is stabilized by hydrogen bonds from His-274 and a water molecule. The conditions for formation of a low-barrier hydrogen bond do not appear to be met, and the calculated hydrogen bond stabilization in the reaction is less than the gas-phase energy, due to interactions with Asp-375 at the active site. The enolate character of the intermediate is apparently necessary for the condensation reaction to proceed efficiently.
...
PMID:Acetyl-CoA enolization in citrate synthase: a quantum mechanical/molecular mechanical (QM/MM) study. 903 8

The effect of gonadectomy on lymphocyte proliferation and macrophage function (hydrogen peroxide production and phagocytosis capacity) of male and female rats was examined and the results correlated with the activities of hexokinase, glucose-6-phosphate dehydrogenase, citrate synthase and phosphate-dependent glutaminase. Also, the reversion of the changes by the treatment with oestrogen or progesterone or a combination of both was addressed. Taken as a whole, ovariectomy reduced hydrogen peroxide production and phagocytosis capacity by macrophages and also lymphocyte proliferation. Castration of male rats reduced the proliferation of lymphocytes and raised macrophage phagocytosis capacity. The effects on macrophage function were correlated with changes in glucose metabolism, particularly, in the activity of glucose-6-phosphate dehydrogenase.
...
PMID:Gonadectomy impairs lymphocyte proliferation and macrophage function in male and female rats. Correlation with key enzyme activities of glucose and glutamine metabolism. 941 77

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.
...
PMID:The effect of the intersubunit disulfide bond on the structural and functional properties of the small heat shock protein Hsp25. 965 71

This work reports the relative importance of the interactions provided by three catalytic residues to individual steps in the mechanism of citrate synthase. When the side chains of any of the residues (H320, D375, and H274) are mutated, the data indicate that they are involved in the stabilization of one or more of the transition/intermediate states in the multistep citrate synthase reaction. H320 forms a hydrogen bond with the carbonyl of oxaloacetate and the alcohols of the citryl-coenzyme A and citrate products. Enzymes substituted at H320 (Q, G, N, and R) have reaction profiles for which the condensation reaction is cleanly rate determining. None of these mutants can activate the carbonyl of oxaloacetate by polarization. All these mutants catalyze the necessary proton transfer from the methyl group of acetyl-coenzyme A only poorly, a process which occurs in a structurally separate site. Furthermore, all H320 mutants hydrolyze the citryl-coenzyme A intermediate significantly more slowly than does the wild-type. D375 is the base removing the proton of acetyl-coenzyme A. D375E and D375G have greatly diminished ability to catalyze proton transfer from acetyl-CoA. The D375 mutants polarize the oxaloacetate carbonyl as well as wild-type. For D375E, the hydrolysis of citryl-CoA is rate determining. D375G, having no side chain capable of acid-base chemistry in either the condensation or hydrolysis reactions is nearly completely devoid of activity in any of the reactions catalyzed by the wild-type. H274 hydrogen bonds to the carbonyl of acetyl-coenzyme A but also forms the back wall of the oxaloacetate-binding site. H274G cannot properly activate either oxaloacetate or acetyl-coenzyme A, and the condensation reaction is overwhelmingly rate determining. Nonetheless, hydrolysis of the intermediate is impaired. All the enzymes except H320R and H274G show kinetic cooperativity with CitCoA as substrate, indicating changes in the subunit interactions with these latter two mutants. The energetics of citrate synthase are surprisingly tightly coupled. All changes affect more than one step in the catalytic cycle. Within the condensation reaction, the intermediate of proton transfer must occupy a shallow well between transition states close in free energy so that perturbations of one have substantial effects on that of the other.
...
PMID:Effects of changes in three catalytic residues on the relative stabilities of some of the intermediates and transition states in the citrate synthase reaction. 965 85

Drosophila melanogaster displays an age-associated increase in oxidative damage and a decrease in mitochondrial transcripts. To determine if these changes result in energy production deficiencies, we measured the electron transport system (ETS) enzyme activity, and ATP levels with age. No statistically significant influences of age on activities of complexes I and II or citrate synthase were observed. In contrast, from 2 to 45 days post-eclosion, declines were found in complex IV cytochrome c oxidase activity (COX, 40% decline) and ATP abundance (15%), while lipid peroxidation increased 71%. We next examined flies that were either genetically or chemically oxidatively stressed to determine the effect on levels of mitochondrial-encoded cytochrome oxidase I RNA (coxI) and COX activity. A catalase null mutant line had 48% of coxI RNA compared to the wild type. In Cu/Zn superoxide dismutase (cSOD) null flies, the rate of coxI RNA decline was greater than in controls. CoxI RNA also declined with increasing hydrogen peroxide (H2O2) treatment, which was reflected in reduced cytochrome c oxidase (COX) activity. These results show that oxidative stress is closely associated with reductions in mitochondrial transcript levels and support the hypothesis that oxidative stress may contribute to mitochondrial dysfunction and aging in D. melanogaster.
...
PMID:Oxidative stress and aging reduce COX I RNA and cytochrome oxidase activity in Drosophila. 980 Oct 75


<< Previous 1 2 3 4 5 6 7 Next >>

---