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)

Short, strong (low barrier) hydrogen bonds occur when the pK values of the atoms sharing the proton are similar. The overall distance is 2.5 A or less, the deuterium fractionation factor is less than 0.5, the proton NMR chemical shift can approach 20 ppm, and deuterium or tritium substitution causes an up-field change in the chemical shift. Such bonds can have deltaH values of 25 kcal/mol in the gas phase, and at least half that in water or other high-dielectric medium. The strength of the hydrogen bond in an active site drops by approximately 1 kcal/mol for each pH unit mismatch in pKs. When a weak hydrogen bond in the initial enzyme-substrate complex is converted into a low-barrier one by alteration of the pK of the substrate or catalytic group so that the pKs match, the increase in hydrogen bond strength can be used to help catalyze the reaction. A well-established example of this is the reaction catalyzed by serine proteases. The pK of neutral histidine is 14, while that of aspartate is approximately 6. Proton transfer from serine to permit attack on bound substrate produces protonated histidine, with a pK now matching that of aspartate. Studies with trifluoromethyl ketone inhibitors that form tetrahedral adducts show up to five orders of magnitude in binding strength as the result of formation of a low-barrier hydrogen bond between aspartate and histidine. Other enzymes whose mechanisms appear to involve low-barrier hydrogen bonds include liver alcohol dehydrogenase, steroid isomerase, triose-P isomerase, aconitase, citrate synthase, and zinc proteases. It is likely that low-barrier hydrogen bonds form at the transition state of any reaction involving general-acid or general-base catalysis, as at that point the pKs of the catalytic group and reactant will be equal.
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PMID:Low-barrier hydrogen bonds and enzymatic catalysis. 1105 Oct 90

The fatty acid elongase-1 beta-ketoacyl-CoA synthase, FAE1 KCS, a seed-specific elongase condensing enzyme from Arabidopsis, is involved in the production of eicosenoic (C20:1) and erucic (C22:1) acids. Alignment of the amino acid sequences of FAE1 KCS, KCS1, and five other putative elongase condensing enzymes (KCSs) revealed the presence of six conserved cysteine and four conserved histidine residues. Each of the conserved cysteine and histidine residues was individually converted by site-directed mutagenesis to both alanine and serine, and alanine and lysine respectively. After expression in yeast cells, the mutant enzymes were analyzed for their fatty acid elongase activity. Our results indicated that only cysteine 223 is an essential residue for enzyme activity, presumably for acyl chain transfer. All histidine substitutions resulted in complete loss of elongase activity. The loss of activity of these mutants was not due to their lower expression level since immunoblot analysis confirmed each was expressed to the same extent as the wild type FAE1 KCS.
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PMID:Active-site residues of a plant membrane-bound fatty acid elongase beta-ketoacyl-CoA synthase, FAE1 KCS. 1134 60

To examine functions of two small heat shock proteins of Escherichia coli, IbpA and IbpB, we constructed His-IbpA and His-IbpB, in which a polyhistidine tag was fused to the N-terminals. Both purified His-IbpA and His-IbpB formed multimers, which have molecular masses of about 2.0-3.0 MDa and consist of about 100-150 subunits. They suppressed the inactivation of several enzymes including citrate synthase and 6-phosphogluconate dehydrogenase by heat, potassium superoxide, hydrogen peroxide and freeze-thawing, but not the inactivation of glyceraldehyde-3-phosphate dehydrogenase by hydrogen peroxide. Both His-IbpA and His-IbpB suppressed enzyme inactivation by various treatments and were also found to be associated with their non-native forms. However, both His-IbpA and His-IbpB were not able to reactivate enzymes inactivated by heat, oxidants or guanidine hydrochloride. When heated to 50 degrees C, each multimeric form of His-IbpA or His-IbpB was dissociated to form a monomer for His-IbpA, and an oligomer of about one-quarter size for His-IbpB. These structural changes were reversible, as both heated proteins regained the multimeric structures after incubation at 25 degrees C. However, when exposed to hydrogen peroxide or potassium superoxide, the large multimeric forms of His-IbpA and His-IbpB were maintained. The results suggest that His-IbpA and His-IbpB suppress the inactivation of enzymes and bind non-native proteins to protect their structures from heat and oxidants.
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PMID:Escherichia coli small heat shock proteins, IbpA and IbpB, protect enzymes from inactivation by heat and oxidants. 1207 54

To clarify the importance of deleted protein and tRNA genes on the impairment of mitochondrial function, we performed a quantitative analysis of biochemical, genetic and morphological findings in skeletal muscles of 16 patients with single deletions and 5 patients with multiple deletions of mtDNA. Clinically, all patients showed chronic progressive external ophthalmoplegia (CPEO). The size of deletions varied between 2.5 and 9 kb, and heteroplasmy between 31% and 94%. In patients with single deletions, the citrate synthase (CS) activity was nearly doubled. Decreased ratios of pyruvate- and succinate-dependent respiration were detected in fibers of all patients in comparison to controls. Inverse and linear correlations without thresholds were established between heteroplasmy and (i) CS referenced activities of the complexes of respiratory chain, (ii) CS referenced maximal respiratory rates, (iii) and cytochrome-c-oxidase (COX) negative fibers. In patients with single and multiple deletions, all respiratory chain complexes as well as the respiratory rates were decreased to a similar extent. All changes detected in patients with single deletions were independent of deletion size. In one patient, only genes of ND5, ND4L as well as tRNA(Leu(CUN)), tRNA(Ser(AGY)), and tRNA(His) were deleted. The pronounced decrease in COX activity in this patient points to the high pathological impact of these missing tRNA genes. The activity of nuclear encoded SDH was also significantly decreased in patients, but to a lesser extent. This is an indication of secondary disturbances of mitochondria at CPEO. In conclusion, we have shown that different deletions cause mitochondrial impairments of the same phenotype correlating with heteroplasmy. The missing threshold at the level of mitochondrial function seems to be characteristic for large-scale deletions were tRNA and protein genes are deleted.
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PMID:Mitochondrial respiratory rates and activities of respiratory chain complexes correlate linearly with heteroplasmy of deleted mtDNA without threshold and independently of deletion size. 1235 Dec 17

A beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III; short-chain condensing enzyme) has been partly purified from pea leaves. The enzyme, which had acetyl-CoA:ACP acyltransferase (ACAT) activity, was resolved from a second, specific, ACAT protein. The KAS III enzyme had a derived molecular mass of 42 kDa (from its cDNA sequence) and operated as a dimer. Its enzymological characteristics were similar to those of two other plant KAS III enzymes except for its inhibition by thiolactomycin. A derivative of thiolactomycin containing a longer (C8 saturated) hydrophobic side-chain (compound 332) was a more effective inhibitor of pea KAS III and showed competitive inhibition towards malonyl-ACP whereas thiolactomycin showed uncompetitive characteristics at high concentrations. This difference may be due to the better fit of compound 332 into a hydrophobic pocket at the active site. A full-length cDNA for the pea KAS III was isolated. This was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in order to facilitate subsequent purification. Demonstrated activity in preparations from E. coli confirmed that the cDNA encoded a KAS III enzyme. Furthermore, the expressed KAS III had ACAT activity, showing that the latter was inherent. The derived amino acid sequence of the pea cDNA showed 81-87% similarity to that for other plant dicotyledon KAS IIIs, somewhat less for Allium porrum (leek, 71%) and for Porphyra spp. (62%), Synechocystis spp. (65%) and various bacteria (42-65%). The pea KAS III exhibited four areas of homology, three of which were around the active-site Cys(123), His(323) and Asn(353). In addition, a stretch of 23 amino acids (residues 207-229 in the pea KAS III) was almost completely conserved in the plant KAS IIIs. Modelling this stretch showed they belonged to a peptide fragment that fitted over the active site and contained segments suggested to be involved in substrate binding and in conformational changes during catalysis, as well as an arginine suggested to participate in the acid-base catalytic mechanism.
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PMID:Beta-ketoacyl-acyl carrier protein synthase III from pea (Pisum sativum L.): properties, inhibition by a novel thiolactomycin analogue and isolation of a cDNA clone encoding the enzyme. 1262 62

Substrate specificity of condensing enzymes is a predominant factor determining the nature of fatty acyl chains synthesized by type II fatty acid synthase (FAS) enzyme complexes composed of discrete enzymes. The gene (mtKAS) encoding the condensing enzyme, beta-ketoacyl-[acyl carrier protein] (ACP) synthase (KAS), constituent of the mitochondrial FAS was cloned from Arabidopsis thaliana, and its product was purified and characterized. The mtKAS cDNA complemented the KAS II defect in the E. coli CY244 strain mutated in both fabB and fabF encoding KAS I and KAS II, respectively, demonstrating its ability to catalyze the condensation reaction in fatty acid synthesis. In vitro assays using extracts of CY244 containing all E. coli FAS components, except that KAS I and II were replaced by mtKAS, gave C(4)-C(18) fatty acids exhibiting a bimodal distribution with peaks at C(8) and C(14)-C(16). Previously observed bimodal distributions obtained using mitochondrial extracts appear attributable to the mtKAS enzyme in the extracts. Although the mtKAS sequence is most similar to that of bacterial KAS IIs, sensitivity of mtKAS to the antibiotic cerulenin resembles that of E. coli KAS I. In the first or priming condensation reaction of de novo fatty acid synthesis, purified His-tagged mtKAS efficiently utilized malonyl-ACP, but not acetyl-CoA as primer substrate. Intracellular targeting using green fluorescent protein, Western blot, and deletion analyses identified an N-terminal signal conveying mtKAS into mitochondria. Thus, mtKAS with its broad chain length specificity accomplishes all condensation steps in mitochondrial fatty acid synthesis, whereas in plastids three KAS enzymes are required.
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PMID:Identification and molecular characterization of the beta-ketoacyl-[acyl carrier protein] synthase component of the Arabidopsis mitochondrial fatty acid synthase. 1466 Jun 74

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS), EC 4.1.3.5, is an essential enzyme in rubber biosynthesis in Hevea brasiliensis. We have isolated a new cDNA encoding HMGS in H. brasiliensis. The full-length hmgs2 consists of 1,916-bp and encodes a protein of 464 amino acids with a predicted molecular mass of 51.27 kDa and an isoelectric point of 6.02. In comparison, HMGS1 and HMGS2 show 92% and 94% nucleotide and amino acid sequence identities, respectively. Semiquantitative RT-PCR analysis indicates that the hmgs2 is more highly expressed in laticifer and petiole than in leaves. Sequence searching and alignment revealed that HMGS is a distant relative of the condensing enzyme; beta-ketoacyl acyl carrier protein synthase III (ACP synthase III), EC 2.3.1.41, identified three completely conserved residues; Cys(117), His(247), and Asn(326). The relationship was greatly strengthened by making a proper alignment of numerous sequences of both HMGS and ACP synthase III. The same Cys(117), His(247), and Asn(326) absolutely conserved in both groups play a catalytic role in ACP synthase III, while such a role of Cys and His has only been reported for HMGS. According to site-directed mutagenesis, the expressed wild-type enzyme shows comparable level with mutant proteins. The mutation of Cys(117) and Asn(326) affects the HMGS activity, indicating that Cys(117) and Asn(326) are important amino acids for the catalytic activity of HMGS. A phylogenetic tree constructed on the basis of proper multiple alignment indicates that HMGS1 and HMGS2 result from recent gene duplication. This is also the case for HMGS and ACP synthase III, which appear to have arisen from an ancient gene duplication event of an ancestral condensing enzyme. Therefore, a possible secondary structure of HMGS could be predicted based on the Protein Data Bank information of ACP synthase III.
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PMID:Molecular cloning of a new cDNA and expression of 3-hydroxy-3-methylglutaryl-CoA synthase gene from Hevea brasiliensis. 1574 97

Knowledge of the interactive domains on the surface of small heat shock proteins (sHSPs) is necessary for understanding the assembly of complexes and the activity as molecular chaperones. The primary sequences of 26 sHSP molecular chaperones were aligned and compared. In the interactive beta3 sequence, 73DRFSVNLDVKHFS85 of human alphaB crystallin, Ser-76, Asn-78, Lys-82, and His-83 were identified as nonconserved residues on the exposed surface of the alpha crystallin core domain. Site-directed mutagenesis produced the mutant alphaB crystallins: S76E, N78G, K82Q, and H83F. Domain swapping with homologous beta3 sequences, 32EKFEVGLDVQFFT44 from Caenorhabditis elegans sHSP12.2 or 69DKFVIFLDVKHFS81 from alphaA crystallin, resulted in the mutant alphaB crystallins, CE1 and alphaA1, respectively. Decreased chaperone activity was observed with the point mutants N78G, K82Q, and H83F and with the mutant, CE1, in aggregation assays using betaL crystallin, alcohol dehydrogenase (ADH), or citrate synthase (CS). The S76E mutant had minimal effect on chaperone activity, and domain swapping with alphaA crystallin had no effect on chaperone activity. The mutations that resulted in altered chaperone activity, produced minimal modification to the secondary, tertiary, and quaternary structure of human alphaB crystallin as determined by ultraviolet circular dichroism spectroscopy, chymotrypsin proteolysis, and size exclusion chromatography. Chaperone activity was influenced by the amount of unfolding of the target proteins and independent of complex size. The results characterized the importance of the exposed side chains of Glu-78, Lys-82, and His-83 in the interactive beta3 sequence of the alpha crystallin core domain in alphaB crystallin for chaperone function.
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PMID:The function of the beta3 interactive domain in the small heat shock protein and molecular chaperone, human alphaB crystallin. 1681 25

Ageing is associated with a reduction in muscle carnosine (beta-alanyl-L-histidine), but there are no data on the changes specifically in type I and type II muscle fibres. Given the higher carnosine content of type II fibers, changes observed in whole muscle may be secondary to a shift in fibre composition. Carnosine, beta-alanine, histidine, taurine, and citrate synthase (CS) and glycogen phosphorylase (Phos), were measured in pools of single muscle fibres from freeze-dried muscle biopsies of vastus lateralis of nine elderly sedentary subjects (65-80 years) with osteoarthritis of the knee and undergoing total knee replacement, and nine young moderately active healthy subjects (20-35 years). Fibres were characterised as type I or II by myosin ATPase activity. Carnosine was 53.2% lower in type II fibres of older subjects resulting in an estimated 7% (and most probably still higher) decline in intracellular physico-chemical buffering capacity. Younger subjects showed higher CS activities in type I and higher Phos activities in type II fibres. These differences were less apparent in elderly subjects. Possible causes for the change in the carnosine content are reduced physical activity, reduced meat intake, or the result of progressive denervation.
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PMID:Carnosine, taurine and enzyme activities of human skeletal muscle fibres from elderly subjects with osteoarthritis and young moderately active subjects. 1696 7

The gene encoding the small heat shock protein (sHSP), LeHSP21.5, has been previously cloned from tomato (GenBank accession no. AB026983). The deduced amino acid sequence of this tomato sHSP was most similar to that of other endoplasmic reticulum (ER)-localized sHSPs (ER-sHSP) and can be predicted to target the ER. We examined whether the gene product of LeHSP21.5 (probable ER-sHSP) can act as molecular chaperone. For functional analysis, LeHSP21.5 protein was expressed in Escherichia coli as His(6)-tagged protein in the C-terminal and purified. We confirmed that ER-sHSP could provide thermal protection of soluble proteins in vitro. We compared the thermal stability of E. coli strain BL21 (DE3) transformed with pET-ER-sHSP with the control E. coli strain BL21(DE3) transformed with only the pET vector under heat shock and IPTG-induced conditions. Most of the protein extracts from E. coli cells expressing ER-sHSP were protected from heat-induced denaturation, whereas extracts from cells not expressing ER-sHSP were very heat-sensitive under these conditions. A similar protective effect was observed when purified ER-sHSP was added to an E. coli cell extract. ER-sHSP prevented the thermal aggregation and inactivation of citrate synthase. These collective findings indicate that ER-sHSP can function as a molecular chaperone in vitro.
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PMID:Molecular chaperone activity of tomato (Lycopersicon esculentum) endoplasmic reticulum-located small heat shock protein. 1828 62


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