Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT00572 (Asn)
 11,732 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transaminase B (branched-chain amino acid aminotransferase, EC 2.6.1.42), the ilvE gene product, was purified to apparent homogeneity from an Escherichia coli K-12 strain which carries the ilvE gene both on the host chromosome and on a plasmid. The oligomeric structure of the enzyme, as determined by analytical ultracentrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was confirmed to be that of a hexamer with a molecular weight of about 182,000 and apparently identical subunits. Cross-linking with dimethylsuberimidate yielded trimers, dimers, and monomers, but essentially no species of higher molecular weight. These results are consistent with a double-trimer arrangement of the subunits in native enzyme. The amino-terminal sequence was found to be: Gly Thr Lys Lys Ala Asp Tyr Ile (Trp) Phe Asn Gly (Thr) (Met) Val. Purified transaminase B catalyzed transamination between alpha-ketoglutarate and l-isoleucine, l-leucine, l-valine, and, to a lesser extent, l-phenylalanine and l-tyrosine, the latter reacting very sluggishly. The enzyme was free of aspartate transaminase and of transaminase C. The apparent K(m) values for the branched-chain alpha-ketoacids were smaller than those for the corresponding amino acids. The lowest K(m) was recorded for dl-alpha-keto-beta-methyl-n-valerate, and the highest was recorded for l-valine. The ratio of the valine- and isoleucine-alpha-ketoglutarate activities did not change significantly during purification, and both activities were quantitatively removed from crude extract by antibody raised against purified transaminase B. These observations argue against the existence of a separate valine-alpha-ketoglutarate transaminase. Anti-E. coli transaminase B antibody cross-reacted with crude extract from Salmonella typhimurium, but not with extract obtained from Pseudomonas aeruginosa.
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PMID:Transaminase B from Escherichia coli: quaternary structure, amino-terminal sequence, substrate specificity, and absence of a separate valine-alpha-ketoglutarate activity. 37 64

The pyridoxal form of both cytosolic and mitochondrial aspartate aminotransferase is irreversibly inactivated consequent to its interaction with the beta,gamma-unsaturated substrate analogue vinylglycine. Per catalytic cycle, 90% of the enzyme molecules are inactivated while 10% escape inactivation by transamination to the pyridoxamine form. In the presence of vinylglycine plus 2-oxoglutarate, inactivation is complete because of retransamination of the pyridoxamine form to the susceptible pyridoxal form. Peptide analyses after inactivation with [1-14C]vinylglycine showed that vinylglycine alkylates the active-site lysine residue 258 which forms the internal aldimine with the coenzyme pyridoxal 5'-phosphate. The coenzyme itself is left intact; resolution of the inactivated enzyme by base or trichloroacetic acid yields pyridoxal-5'-P. The absorption spectrum of the inactivated enzyme (lambdamax 335 nm) suggests that the cofactor is bound as a substituted aldimine. The proposed pathway of alkylation of Lys-258 involves abstraction of the alpha proton from vinylglycine, isomerization to the alpha,beta-unsaturated enamine, and subsequent nucleophilic attack of the epsilon-amino group of the lysyl residue at the beta carbon of the inhibitor. The determination of the amino acid sequence around the coenzyme-binding lysyl residue in the mitochondrial isoenzyme from chicken gave Ala-(epsilon-Pxy)Lys-Asn-Met-(Gly,Leu,Tyr) which is identical with the other mitochondrial transaminases examined so far.
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PMID:Active-site labeling of aspartate aminotransferases by the beta,gamma-unsaturated amino acid vinylglycine. 91 93

The sequences of the coenzyme-binding peptide of both cytoplasmic and mitochondrial aspartate aminotransferases from sheep liver were determined. The holoenzymes were treated with NaBH4 and digested with chymotrypsin; peptides containing bound pyridoxal phosphate were then isolated. One phosphopyridoxyl peptide was obtained from sheep liver cytoplasmic aspartate aminotransferase. Its sequence was Ser-Ne-(phosphopyridoxyl)-Lys-Asn-Phe. This sequence is identical with that reported for the homologous peptide from pig heart cytoplasmic aspartate aminotransferase. Two phosphopyridoxyl peptides with different RF values were isolated from the sheep liver mitochondrial isoenzyme. They had the same N-terminal amino acid and similar amino acid composition. The mitochondrial phosphopyridoxyl peptide of highest yield and purity had the sequence Ala-Ne-(phosphopyridoxyl)-Lys-Asx-Met-Gly-Leu-Tyr. The sequence of the first four amino acids is identical with that already reported for the phosphopyridoxyl tetrapeptide from the pig heart mitochondrial isoenzyme. The heptapeptide found for the sheep liver mitochondrial isoenzyme closely resembles the corresponding sequence taken from the primary structure of the pig heart cytoplasmic aspartate aminotransferase.
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PMID:The sequences of the coenzyme-binding peptide in the cytoplasmic and the mitochondrial aspartate aminotransferases from sheep liver. 118 Aug 94

In four abnormal fibrinogens with a point mutation in the gamma chain, all characterized by impaired fibrin polymerization, we identified single base exchanges in the respective mutant gamma chain genes by polymerase chain reaction followed by DNA sequence analysis. These base exchanges accounted for the amino acid substitutions previously reported from our laboratory. They were exchanges of C to T (CGC for gamma Arg-275 to TGC for Cys) in fibrinogen Osaka II, T to G (AAT for gamma Asn-308 to AAG for Lys) in fibrinogen Kyoto I, T to C (ATG for gamma Met-310 to ACG for Thr) in fibrinogen Asahi, and G to T (GAT for gamma Asp-330 to TAT for Tyr) in fibrinogen Kyoto III. These base exchanges were found to reside in exon VIII of the gamma chain gene. Since many abnormal molecules are associated with polymerization defects, unless associated with the impaired release of fibrinopeptides A and/or B, exon VIII of the gamma chain gene may deserve careful study to define the structural alterations.
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PMID:Gene analyses of abnormal fibrinogens with a mutation in the gamma chain. 142 Nov 74

Human liver cholesterol 7 alpha-hydroxylase (CYP7) cDNAs were isolated from a human liver cDNA library. A full-length cDNA has 2901 nucleotides which encode a typical P450 polypeptide of 504 amino acid residues. Two different sequences of codon 100, TTT (Phe) and TCT (Ser), were identified in cDNA clones. In addition, codons 347 and 385 are GAT (Asp) and GAC (Asp) in all cDNA clones, whereas those reported previously (FEBS Lett. 268, 137-140, 1990) are AAT (Asn) and AGC (Ser), respectively. Since there is only one 7 alpha-hydroxylase gene in the human genome, it is likely that polymorphisms at the codon 100 of cDNA clones arise from two different alleles in the 7 alpha-hydroxylase gene of this human liver.
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PMID:Polymorphisms of human cholesterol 7 alpha-hydroxylase. 161 Mar 52

Asp222 is an invariant residue in all known sequences of aspartate aminotransferases from a variety of sources and is located within a distance of strong ionic interaction with N(1) of the coenzyme, pyridoxal 5'-phosphate (PLP), or pyridoxamine 5'-phosphate (PMP). This residue of Escherichia coli aspartate aminotransferase was replaced by Ala, Asn, or Glu by site-directed mutagenesis. The PLP form of the mutant enzyme D222E showed pH-dependent spectral changes with a pKa value of 6.44 for the protonation of the internal aldimine bond, slightly lower than that (6.7) for the wild-type enzyme. In contrast, the internal aldimine bond in the D222A or D222N enzyme did not titrate over the pH range 5.3-9.5, and a 430-nm band attributed to the protonated aldimine persisted even at high pH. The binding affinity of the D222A and D222N enzymes for PMP decreased by 3 orders of magnitude as compared to that of the wild-type enzyme. Pre-steady-state half-transamination reactions of all the mutant enzymes with substrates exhibited anomalous progress curves comprising multiphasic exponential processes, which were accounted for by postulating several kinetically different enzyme species for both the PLP and PMP forms of each mutant enzyme. While the replacement of Asp222 by Glu yielded fairly active enzyme species, the replacement by Ala and Asn resulted in 8600- and 20,000-fold decreases, respectively, in the catalytic efficiency (kmax/Kd value for the most active species of each mutant enzyme) in the reactions of the PLP form with aspartate. In contrast, the catalytic efficiency of the PMP form of the D222A or D222N enzyme with 2-oxoglutarate was still retained at a level as high as 2-10% of that of the wild-type enzyme. The presteady-state reactions of these two mutant enzymes with [2-2H]aspartate revealed a deuterium isotope effect (kH/kD = 6.0) greater than that [kH/kD = 2.2; Kuramitsu, S., Hiromi, K., Hayashi, H., Morino, Y., & Kagamiyama, H. (1990) Biochemistry 29, 5469-5476] for the wild-type enzyme. These findings indicate that the presence of a negatively charged residue at position 222 is particularly critical for the withdrawal of the alpha-proton of the amino acid substrate and accelerates this rate-determining step by about 5 kcal.mol-1. Thus it is concluded that Asp222 serves as a protein ligand tethering the coenzyme in a productive mode within the active site and stabilizes the protonated N(1) of the coenzyme to strengthen the electron-withdrawing capacity of the coenzyme.
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PMID:Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate. 161 Aug 31

In aspartate aminotransferase (AspAT), His143 is located within a hydrogen-bonding distance to Asp222 that forms a strong ion pair with the ring nitrogen of the coenzyme, pyridoxal 5'-phosphate (PLP) or pyridoxamine 5'-phosphate (PMP). His143 of Escherichia coli AspAT was replaced by Ala or Asn. The mutant enzyme H143A showed a slight increase in the maximum velocity of the overall transamination reaction between aspartate and 2-oxoglutarate, while H143N AspAT showed a decrease to 60% in the maximum rate of the overall reactions in both directions. In all of the half-transamination reactions with four substrates, aspartate, glutamate, oxalacetate, and 2-oxoglutarate, the catalytic competence as defined by kmax/Kd decreased by 3-18-fold upon replacing His143 by either Ala or Asn. The extent of the decrease varied from one substrate to another; it was largely contributed to by the decrease in affinities for all substrates. The equilibrium constants, [PMP-form] [keto acid]/[( PLP-form] [amino acid]), decreased by over 10-fold upon the mutations at position 143. Both H143A and H143N AspATs exhibited a considerably decreased affinity for 2-methylaspartate, an external-aldimine-forming substrate analogue, yet without appreciable alteration in the affinity for succinate and glutarate, which are non-aldimine-forming analogues. All these findings suggest that, although His143 is not essential for catalysis, it might assist the formation of enzyme-substrate complex.
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PMID:The role of His143 in the catalytic mechanism of Escherichia coli aspartate aminotransferase. 200 66

This work was undertaken as part of a search for well-characterized glycoprotein models in which both the oligosaccharide structure, the number of oligosaccharide chains, and the precise location of these chains in the protein are known. On the basis of the fact that high-affinity ligand binding sites have been defined precisely for several proteins in terms of both number and relative location, the hypothesis to be tested was that if oligosaccharide chains were covalently attached to such high-affinity ligands, they would be specifically bound in the ligand sites of the appropriate protein, thus permitting the preparation of neoglycoproteins of precise predetermined oligosaccharide valency and topography. To test this hypothesis, pyridoxal 5'-phosphate was reductively (NaB3H4) aminated with the alpha-amino group of the asparagine oligosaccharide Man6-GlcNAc2-Asn from ovalbumin. When the resulting phosphopyridoxylated oligosaccharide (PG) was added to the apo form of aspartate aminotransferase (AAT; EC 2.6.1.1, the cytosolic enzyme from pig heart, consisting of two subunits and containing two coenzyme binding sites), a 2:1 (PG-AAT) complex was formed which could be characterized on the basis of tritium content, the absorbance and fluorescence of the pyridoxamine phosphate moiety of PG, and the concanavalin A binding properties acquired by AAT through the incorporation of the oligosaccharide. As expected from the established properties of the holoenzyme, the AAT-PG complex is stable in the absence of phosphate or vitamin B6 derivatives and can be dialyzed for 24 h without any significant loss of PG. According to the three-dimensional model of AAT, the oligosaccharide chain of PG should be partially masked in the coenzyme binding pocket.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Neoglycoproteins: preparation of noncovalent glycoproteins through high-affinity protein- (glycosyl) ligand complexes. 646 43

Alleles of the var1 locus on yeast mitochondrial DNA specify the size of var1 ribosomal protein. We report the nucleotide sequence of a var1 allele that determines the smallest var1 protein. It contains an open reading frame of 396 codons, which we identify as the structural gene for var1 protein. The var1 protein specified by this allele has an amino acid composition in close agreement with that predicted by the DNA sequence. The var1 coding region is highly unusual: it is 89.6% AT and contains a 46 bp GC-rich palindromic cluster that accounts for 38% of the total GC residues. Our results strongly suggest that like mammalian mitochondria but unlike those from Neurospora, yeast mitochondria use AUA as a methionine codon. Comparison with the sequence of a var1 allele specifying a larger protein suggests that some size polymorphism of var1 protein results from in-frame insertions of a variable number of AAT (Asn) codons.
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PMID:Location and structure of the var1 gene on yeast mitochondrial DNA: nucleotide sequence of the 40.0 allele. 675 87

The complete amino acid sequence of the hen ovomacroglobulin (ovostatin) subunit has been determined from cDNA and partial peptide sequence analysis. Ovostatin is a tetrameric member of the alpha-macroglobulin (alpha M) family of proteins. The 4715 nt ovostatin cDNA encodes a 36- or a 16-residue signal peptide and a 1437-residue mature protein (162.2 kDa). At the protein level the overall score of sequence identity between ovostatin and mammalian alpha Ms is 39-44%, indicating an early divergence from the line leading to the mammalian alpha Ms. Ovostatin contains 56 mol glucosamine per mol subunit, and 12 of its Asn-residues are likely to be N-glycosylated. Including carbohydrate, the size of the ovostatin subunit is approx. 185 kDa. The ovostatin subunit is predicted to contain 12 intrachain disulfide bridges, and two subunits are predicted to be disulfide bound by two interchain bridges. One Cys residue may be unpaired or participate in dimer formation as a third interchain disulfide bridge. Ovostatin contains a unique 40-residue bait region. In contrast to other alpha Ms, ovostatin contains no internal beta-Cys-gamma-Glu thiol ester, as a result of a Cys-to-Asn replacement (TGC or TGT to AAT), but the Gln-moiety of the thiol ester is preserved. By comparing the sequences of the receptor binding domain in alpha Ms with the corresponding region of ovostatin possible determinants for receptor recognition of mammalian alpha Ms are proposed.
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PMID:Amino acid sequence of hen ovomacroglobulin (ovostatin) deduced from cloned cDNA. 753 98


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