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)

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.
...
PMID:The effect of replacing the conserved active-site residues His-264, Asp-312 and Arg-314 on the binding and catalytic properties of Escherichia coli citrate synthase. 801 Sep 58

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

Rat carnitine palmitoyltransferase (CPT) II was expressed in Saccharomyces cerevisiae. Mitochondrial fractions prepared from the cells displayed high CPT activity and reacted with an antibody to the rat protein on immunoblots, whereas no activity or immunoreactive protein was observed in control cells. The recombinant enzyme was largely membrane associated. Treatment of the expressed protein with diethyl pyrocarbonate, a reagent that modifies histidine residues, abolished CPT activity, but this was completely restored by reversal of the modification with hydroxylamine. It is inferred that a histidine residue plays a critical role in CPT function. Expression and analysis of site-directed mutants of CPT II showed that histidine 372, as well as aspartates 376 and 464 (all conserved throughout the carnitine/choline acyltransferase family), are essential for catalytic activity. The data suggest that the mechanism by which CPT II effects transesterification between palmitoyl-CoA and carnitine possibly involves histidine 372 and one of these aspartate residues, interacting with the carnitine hydroxyl group, in a reaction analogous to that carried out by a histidine/aspartate/serine catalytic triad in certain other enzyme systems. Mutagenic analysis of a region of CPT II that is highly conserved among the carnitine and choline acyltransferases, and which is homologous to the "adenine binding loop" of citrate synthase, implies that it has no similar function in CPT II.
...
PMID:Catalytically important domains of rat carnitine palmitoyltransferase II as determined by site-directed mutagenesis and chemical modification. Evidence for a critical histidine residue. 803 73

We report properties of five active site mutants of Escherichia coli citrate synthase, in which histidine 264, aspartate 362, and phenylalanine 383 were replaced by alanines, and arginines 387 and 407 by leucines. All mutants have much lower turnover numbers than wild type enzyme; the strongest effect was with the arginine 387 mutant, perhaps because the substrate, oxaloacetate, binds in a different orientation. The arginine 407 mutant has lost most of its ability to distinguish alpha-ketoglutarate, a competitive inhibitor, from oxaloacetate. The mutations of histidine 264 and aspartate 362 affect steady-state kinetics as would be anticipated from current models for citrate synthase catalysis, and resemble mutations of these residues, in pig heart and E. coli enzyme, reported by others. Mutations of residues 264, 362, and 383 also affect allosteric properties. With the phenylalanine 383 mutant, acetyl-CoA saturation is strongly sigmoid, even in the presence of the activator, KCl, implying a marked shift of the allosteric equilibrium toward the T state. The histidine 264 mutant appears to be shifted toward R state and shows weaker binding of the allosteric inhibitor, NADH; thus this mutation also affects the allosteric site, 25-30 A away.
...
PMID:Active site mutants of Escherichia coli citrate synthase. Effects of mutations on catalytic and allosteric properties. 827 29

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

Glyoxysomal malate dehydrogenase (gMDH) is an enzyme of the glyoxylate cycle that participates in degradation of storage oil. We have cloned a cDNA for gMDH from etiolated pumpkin cotyledons that encodes a polypeptide consisting of 356 amino acid residues. The nucleotide and N-terminal amino acid sequences revealed that gMDH is synthesized as a precursor with an N-terminal extrapeptide. The N-terminal presequence of 36 amino acid residues contains two regions homologous to those of other microbody proteins, which are also synthesized as large precursors. To investigate the functions of the N-terminal presequence of gMDH, we generated transgenic Arabidopsis that expressed a chimeric protein consisting of beta-glucuronidase and the N-terminal region of gMDH. Immunological and immunocytochemical studies revealed that the chimeric protein was imported into microbodies such as glyoxysomes and leaf peroxisomes and was then subsequently processed. Site-directed mutagenesis studies showed that the conserved amino acids in the N-terminal presequence, Arg-10 and His-17, function as recognition sites for the targeting to plant microbodies, and Cys-36 in the presequence is responsible for its processing. These results correspond to those from the analyses of glyoxysomal citrate synthase (gCS), which was also synthesized as a large precursor, suggesting that common mechanisms that can recognize the targeting or the processing of gMDH and gCS function in higher plant cells.
...
PMID:Glyoxysomal malate dehydrogenase in pumpkin: cloning of a cDNA and functional analysis of its presequence. 955 62

Chloroplast chaperonin 20 (Cpn20) in higher plants is a functional homologue of the Escherichia coli GroES, which is a critical regulator of chaperonin-mediated protein folding. The cDNA for a Cpn20 homologue of Arabidopsis thaliana was isolated. It was 958 bp long, encoding a protein of 253 amino acids. The protein was composed of an N-terminal chloroplast transit peptide, and the predicted mature region comprised two distinct GroES domains that showed 42% amino acid identity to each other. The isolated cDNA was constitutively expressed in transgenic tobacco. Immunogold labelling showed that Cpn20 is accumulated in chloroplasts of transgenic tobacco. A Northern blot analysis revealed that mRNA for the chloroplast Cpn20 is abundant in leaves and is increased by heat treatment. To examine the oligomeric structure of Cpn20, a histidine-tagged construct lacking the transit peptide was expressed in E. coli and purified by affinity chromatography. Gel-filtration and cross-linking analyses showed that the expressed products formed a tetramer. The expressed products could substitute for GroES to assist the refolding of citrate synthase under non-permissive conditions. The analysis on the subunit stoichiometry of the GroEL-Cpn20 complex also revealed that the functional complex is composed of a GroEL tetradecamer and a Cpn20 tetramer.
...
PMID:Chloroplast Cpn20 forms a tetrameric structure in Arabidopsis thaliana. 1020 3

A unique feature of fatty acid synthase (FAS) type II of higher plants and bacteria is 3-oxoacyl-[acyl-carrier-protein (ACP)] synthase III (KAS III), which catalyses the committing condensing reaction. Working with KAS IIIs from Cuphea seeds we obtained kinetic evidence that KAS III catalysis follows a Ping-Pong mechanism and that these enzymes have substrate-binding sites for acetyl-CoA and malonyl-ACP. It was the aim of the present study to identify these binding sites and to elucidate the catalytic mechanism of recombinant Cuphea wrightii KAS III, which we expressed in Escherichia coli. We engineered mutants, which allowed us to dissect the condensing reaction into three stages, i.e. formation of acyl-enzyme, decarboxylation of malonyl-ACP, and final Claisen condensation. Incubation of recombinant enzyme with [1-(14)C]acetyl-CoA-labelled Cys(111), and the replacement of this residue by Ala and Ser resulted in loss of overall condensing activity. The Cys(111)Ser mutant, however, still was able to bind acetyl-CoA and to catalyse subsequent binding and decarboxylation of malonyl-ACP to acetyl-ACP. We replaced His(261) with Ala and Arg and found that the former lost activity, whereas the latter retained overall condensing activity, which indicated a general-base action of His(261). Double mutants Cys(111)Ser/His(261)Ala and Cys(111)Ser/His(261)Arg were not able to catalyse overall condensation, but the double mutant containing Arg induced decarboxylation of [2-(14)C]malonyl-ACP, a reaction indicating the role of His(261) in general-acid catalysis. Finally, alanine scanning revealed the involvement of Arg(150) and Arg(306) in KAS III catalysis. The results offer for the first time a detailed mechanism for a condensing reaction catalysed by a FAS type II condensing enzyme.
...
PMID:Reaction mechanism of recombinant 3-oxoacyl-(acyl-carrier-protein) synthase III from Cuphea wrightii embryo, a fatty acid synthase type II condensing enzyme. 1060 Jun 51

The mechanism(s) by which heat shock protein 25 (hsp25) protects cells from stress may involve one or more of the biochemical properties attributed to hsp25 and other small M(r) hsp. In this report, structural and functional properties of an N-terminal 33 amino acid deletion variant of hsp25 (termed hsp25.c) were considered by comparison with hsp25. 6-His tagged recombinant hsp25 and hsp25.c (termed (H6)hsp25.a and (H6)hsp25.c) were expressed and purified. Oligomeric proteins formed and possessed properties previously attributed to hsp25. The 33 amino acid deletion represented by hsp27.c did not affect the ability of the recombinant protein to act as an inhibitor of elastase, as a molecular chaperone in the refolding of denatured citrate synthase, or as an actin-binding protein. The overexpression of either hsp25 or hsp25.c, enhanced the stress resistance of stable transformed eukaryotic cells. This N-terminal variant protein may be used in further cellular and biochemical assessment of hsp25 oligomerization and function.
...
PMID:An N-terminal 33-amino-acid-deletion variant of hsp25 retains oligomerization and functional properties. 1073 25

We describe a 17-year-old boy with a clinical neurologic picture consistent with Kearns-Sayre syndrome. His manifestations included progressive external ophthalmoplegia, bilateral ptosis, retinitis pigmentosa, and muscle weakness. He was found to harbor an abundant novel deletion in skeletal muscle mitochondrial DNA. Biochemical analysis of the patient's biopsied skeletal muscle showed that the specific activities of all four respiratory complexes with mitochondrial DNA-encoded subunits were markedly reduced in contrast to normal activity levels of entirely nuclear DNA-encoded enzyme activities (eg, complex II and citrate synthase). Ultrastructural analysis also indicated the presence of strikingly abnormal mitochondria with both unusual cristae and frequent paracrystalline inclusions. The great amount of the deleted mitochondrial DNA in this patient's muscle, as well as the concomitant reduction in specific respiratory complex activity, suggests that the mitochondrial DNA deletion plays a role in the pathogenesis of this neurologic disease.
...
PMID:Kearns-Sayre syndrome with a novel mitochondrial DNA deletion. 1096 96


<< Previous 1 2 3 4 Next >>

---