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:3.4.15.1 (ACE)
18,300 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The aceEF-lpd operon of Escherichia coli encodes the pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase multienzyme complex (PDH complex). A thermoinducible expression system was developed to amplify a variety of genetically restructured PDH complexes, including those containing three, two, one and no lipoyl domains per E2p chain. Although large quantities of the corresponding complexes were produced, they had only 20-50% of the predicted specific activities. The activities of the E1p components were diminished to the same extent, and this could account for the shortfall in overall complex activity. Thermoinduction was used to express a mutant PDH complex in which the putative active-site histidine residue of the E2p component (His-602) was replaced by cysteine in the H602C E2p component. This substitution abolished dihydrolipoamide acetyltransferase activity of the complex without affecting other E2p functions. The results support the view that His-602 is an active-site residue. The inactivation could mean that the histidine residue performs an essential role in the acetyltransferase reaction mechanism, or that the reaction is blocked by an irreversible modification of the cysteine substituent. Complementation was observed between the H602C PDH complex and a complex that is totally deficient in lipoyl domains, both in vitro, by the restoration of overall complex activity in mixed extracts, and in vivo, from the nutritional independence of strains that co-express the two complexes from different plasmids.
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PMID:Overexpression of restructured pyruvate dehydrogenase complexes and site-directed mutagenesis of a potential active-site histidine residue. 220 Dec 86

Genomic clones encompassing the entire gene (PDH alpha) encoding the human pyruvate dehydrogenase alpha-subunit (PDH alpha) have been isolated by screening a leukocyte genomic library in the cloning vector, lambda EMBL4. The PDH alpha gene spans 17082 bp and is composed of eleven exons and ten introns. All intron/exon splice junctions follow the GT/AG rule. A total of seven Alu repeats were found in five introns. The entire nucleotide (nt) sequence of the PDH alpha gene has been determined and typical consensus promoter sequences in the 5'-flanking region were found. The results of primer extension analysis imply that the PDH alpha gene transcription start point (tsp) is a thymine residue 124 bp upstream from the ATG start codon in exon 1. The structural organization and the tsp were compared with the recent report [Maragos et al., J. Biol. Chem. 264 (1989) 12294-12298]. Analysis of the PDH alpha gene resolves existing discrepancies among four published sequences of PDH alpha cDNAs. A 93-bp sequence that was missing in our sequence of cultured foreskin fibroblast PDH alpha cDNA [Koike et al., Proc. Natl. Acad. Sci. USA 85 (1988) 41-45] was identified in the gene as exon 6.
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PMID:Characterization and nucleotide sequence of the gene encoding the human pyruvate dehydrogenase alpha-subunit. 222 43

The pyruvate dehydrogenase complex (PDH complex) of Escherichia coli and its pyruvate dehydrogenase component (E1) are rapidly inactivated by low concentrations of fluoropyruvate in a thiamin pyrophosphate (TPP) dependent process. The inactivation rates for the PDH complex and for its E1 component are similar. Pyruvate protects the PDH complex and the E1 component against inactivation by fluoropyruvate. Dihydrolipoamide protects the E1 component from inactivation. TPP is not covalently bound to the PDH complex or to the E1 component by the inactivating reaction. When [14C]fluoropyruvate is used to inactivate the PDH complex, 14C remains bound to the complex after gel filtration. This bound radioactivity is cleaved from the protein by NH2OH, -OH, and NaBH4 but not by dilute acid. When released by -OH, greater than 90% of the 14C cochromatographs with acetate on DEAE-Sephadex. When released by NaBH4, and 14C is recovered as [14C]ethanol. Colorimetric analysis for sulfhydryl groups on the native E1 component and the inactivated E1 component, using 5,5'-dithiobis(2-nitrobenzoate), reveals that complete inactivation results from covalent modification of 1.37 +/- 0.03 sulfhydryl residues. Fluoropyruvate is known to generate acetyl-TPP at the active site of E1. The available evidence indicates that acetylation of a sulfhydryl group by acetyl-TPP at the active site of the E1 component inactivates the enzyme.
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PMID:Inactivation of the pyruvate dehydrogenase complex of Escherichia coli by fluoropyruvate. 251 3

Rate constants for the hydrolysis of acetyl-TPP were measured between pH values of 2.5 and 7.5 and plotted as log kobs versus pH. The pH-rate profile defined two legs, each with a slope of +1 but separated by a region of decreased slope between pH 4 and pH 6. The rates were insensitive to buffer concentrations. Each leg of the profile reflected specific-base-catalyzed hydrolysis of acetyl-TPP, analogous to the hydrolysis of 2-acetyl-3,4-dimethylthiazolium ion [Lienhard, G.E. (1966) J. Am. Chem. Soc. 88, 5642-5649]. The separation of the two legs of this profile has been shown to be caused by the ionization of a group exhibiting a pKa of 4.73 within acetyl-TPP that is remote from the acetyl group, the amino-pyrimidine ring, which is protonated below pH 4.73. The protonation level of this ring has been shown to control the equilibrium partitioning of acetyl-TPP among its carbinolamine, keto, and hydrate forms. The differential partitioning of these species is a major factor causing the separation between the two legs of the pH-rate profile. The characteristic pH-rate profile and the availability of synthetic acetyl-TPP [Gruys, K.J., Halkides, C.J., & Frey, P.A. (1987) Biochemistry 26, 7575-7585] have facilitated the isolation and identification of [1-14C]acetyl-TPP from acid-quenched enzymatic reaction mixtures at steady states. [1-14C]Acetyl-TPP was identified as a transient species in reactions catalyzed by the PDH complex or the pyruvate dehydrogenase component of the complex (E1). The pH-rate profile for hydrolysis of [1-14C]-acetyl-TPP isolated from enzymatic reactions was found to be indistinguishable from that for authentic acetyl-TPP, which constituted positive identification of the 14C-labeled enzymic species.
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PMID:2-Acetylthiamin pyrophosphate (acetyl-TPP) pH-rate profile for hydrolysis of acetyl-TPP and isolation of acetyl-TPP as a transient species in pyruvate dehydrogenase catalyzed reactions. 260 41

These perspectives from the first International Symposium on Primary Biliary Cirrhosis review recent advances and single out some areas for further enquiry. The latter include frequency and type of associated autoimmune diseases, the existence of clinical subsets of PBC, immunohistochemical analysis of lymphoid infiltrates in the liver, effects of immunosuppressive and other treatment regimens, and models for predicting the optimal time for liver transplantation. The M2 autoantigens have been identified as mitochondrial 2-oxo-acid dehydrogenase enzymes. These include pyruvate dehydrogenase (70-74 kd antigen) and branched chain 2-oxo-acid dehydrogenase and 2-oxo-acid glutaric dehydrogenase (45-52 kd antigens). Each of these enzymes has three subunits, E1 to E3. For PDH, an autoepitope has been identified as a decapeptide containing the attachment site of lipoic acid, an essential cofactor for enzyme activity. Current questions include the degree to which antibodies to PDH, and related enzymes, account for the mitochondrial reactivity defined by immunofluorescence or other procedures, the cell-surface expression of M2 autoantigens, and the significance of the occurrence of nonmitochondrial (such as centromeric) autoantibodies in PBC. The unknown T lymphocyte contribution to the autoimmune response in PBC may involve inducer and effector components. A postulated T-cell autoepitope may be presented, in association with MHC class I or class II molecules, on the surface of biliary epithelial cells. T cell lines from PBC livers removed during transplantation could provide data on the T-lymphocyte contribution to the pathogenesis of PBC.
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PMID:Primary biliary cirrhosis: current knowledge, perspectives, and future directions. 265 6

Several enzymes catalyze reactions that may involve acetylthiamin pyrophosphate (acetyl-TPP) as an intermediate. These enzymes are phosphoketolase, pyruvate oxidase and several pyruvate oxidoreductases. Acetyl-TPP can be synthesized and used as a carrier to analyze quenched reaction mixtures for the presence of [14C]acetyl-TPP. Synthetic acetyl-TPP exhibits unusual chemical properties and a unique pH-rate profile that serves as a powerful means of characterizing [14C]acetyl-TPP that has been isolated from quenched enzymatic reaction mixtures. Using this and other methods, extensive evidence has been obtained for the involvement of acetyl-TPP in certain reactions catalyzed by the pyruvate dehydrogenase complex (PDH complex) of Escherichia coli. Acetyl-TPP is chemically competent as an intermediate in the decarboxylation and dehydrogenation of pyruvate by the PDH complex; and it is transiently formed during the course of this reaction. It may be an enzyme-bound intermediate or it may be in equilibrium with such an intermediate. Acetyl-TPP is very likely to be an intermediate of the phosphoketolase reaction. However, no direct evidence linking it to the phosphoketolase reaction mechanism is yet available. It is unclear whether acetyl-TPP is an intermediate in the pyruvate oxidoreductase reactions. In one example, that of the ketoacid oxidoreductase of Halobacterium halobium, analysis by electron paramagnetic resonance spectroscopy indicates the involvement of a hydroxyethyl-TPP-radical as an intermediate. It is unknown whether the subsequent reaction of this radical with coenzyme A an an oxidized FeS cluster to produce acetyl coenzyme A and the reduced cluster involves the intermediate formation of acetyl-TPP.
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PMID:2-Acetylthiamin pyrophosphate: an enzyme-bound intermediate in thiamin pyrophosphate-dependent reactions. 267 49

Autoantibodies are important diagnostic markers for autoimmune type chronic active hepatitis (AI-CAH) and primary biliary cirrhosis (PBC). At least three subgroups of AI-CAH can be distinguished serologically. Antinuclear antibodies (ANA), smooth muscle antibodies (SMA), and liver membrane autoantibodies (LMA) characterize classical autoimmune type 'lupoid' hepatitis, while liver kidney microsomal (LKM) antibodies identify a second, and antibodies to a soluble liver antigen (anti-SLA), a third subgroup of AI-CAH. Patients with autoimmune type CAH in contrast to patients with virus-induced liver diseases profit from immunosuppressive therapy. PBC is characterized by disease-specific subtypes of antimitochondrial antibodies (AMA). Technical developments, like immunoblotting and molecular cloning, led to a better definition and characterization of autoantibody-antigen systems. Molecular cloning has been successfully applied to identify the main 70 kDa mitochondrial antigen in PBC. This and other mitochondrial autoantigens have been identified as enzymes: E2 component of pyruvate dehydrogenase (PDH-E2) and its component X, branched chain alpha-keto acid dehydrogenase (BCKD-E2), and 2-oxoglutarate dehydrogenase. LKM-1 antigen has been identified as cytochrome P-450 db1, a drug metabolizing enzyme with a known genetic polymorphism. These cloned hepatic autoantigens share some characteristics with other autoantigens: they are enzymes, autoantibodies react with active sites of these enzymes and the autoepitopes are highly conserved. After the identification of these autoepitopes, specific and sensitive diagnostic reagents will become available. B and T cell epitope mapping will help to elucidate whether these autoantibodies are just clinically valuable diagnostic markers or whether they contribute to the immunopathogenesis or help to identify the aetiological agents.
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PMID:Autoantibodies and antigens in liver diseases--updated. 268 96

We investigated the temporal relationship between hepatic glycogen depletion and cardiac and hepatic PDH (pyruvate dehydrogenase complex) activities during the acute phase of starvation. There was a striking correlation between the decline in hepatic glycogen and PDH inactivation during the first 10 h of starvation. Re-feeding after 6 h starvation was associated with complete re-activation of PDH in liver and re-activation to approx. 75% of the fed value in heart, whereas in rats previously starved for 24-48 h re-activation was delayed in liver and diminished in heart. The results are discussed with reference to the fate of dietary carbohydrate after re-feeding.
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PMID:Pyruvate dehydrogenase activities during the fed-to-starved transition and on re-feeding after acute or prolonged starvation. 270 97

Meal-fed rats and rats fed ad libitum had similar rates of hepatic glycogenesis at 60 min after the initiation of re-feeding a chow meal after 22 h starvation, but hepatic PDHa (active form of pyruvate dehydrogenase) activities were 4-fold higher in the meal-fed group. In heart, PDHa activities were 3-fold higher before re-feeding and 2-fold higher after re-feeding in the meal-fed group compared with the group fed ad lib. The blood metabolite profile suggested diminished fat oxidation in starved meal-fed rats and accelerated flux through PDH in meal-fed re-fed rats compared with the group fed ad lib.
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PMID:Comparison of tissue pyruvate dehydrogenase activities on re-feeding rats fed ad libitum or meal-fed rats with a chow-diet meal. 281 70

Pyruvate oxidation by normal intact leukocytes has been systematically studied to define optimal conditions for detection of enzymatic defects in this process. Leukocytes were isolated by dextran sedimentation and lymphocytes by Ficoll centrifugation. Cells were incubated for 2 h with [1-14C]-pyruvate, [2-14C]-pyruvate or [1-14C]-acetate as substrate. The specific oxidative capacity of lymphocytes was almost three times higher than that of granulocytes from the same blood. Oxidation of both pyruvate and acetate was highly dependent on the substrate concentration in the medium reaching a plateau between 0.5 and 1.0 mmol/l. Addition of succinate (1 mmol/l) stimulated oxidation of [1-14C]-pyruvate by 30%. Uncoupling of phosphorylation by addition of carbonyl cyanide chlorophenylhydrazone (CCCP) (0.1 mumol/l) increased oxidation of [1-14C]-pyruvate by 200% and of [1-14C]-acetate by 70%. Addition of CCCP plus succinate caused further stimulation of pyruvate oxidation (+40%), but not of acetate oxidation. It is therefore concluded that: (1) Lymphocytes are better than mixed leukocytes for oxidative studies. (2) Unlabelled substrate should be added at optimal concentrations. (3) The pyruvate dehydrogenase complex is normally only partially active in lymphocytes. (4) Stimulation of oxidation by CCCP greatly enhances the flux through the PDH step thus facilitating the detection of defects in pyruvate oxidation.
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PMID:Pyruvate and acetate oxidation by leukocytes in vitro. Activation of the pyruvate dehydrogenase complex by uncoupling of oxidative phosphorylation. 316 Jan


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